Methods and compositions related to adenoassociated virus-phage particles

ABSTRACT

Embodiments of the invention are generally directed to compositions and methods of delivering one or more transgene to a target cell, such as a tumor cell, in a site-specific manner to achieve enhanced expression and to constructs and compositions useful in such applications. In certain aspects, expression from a therapeutic nucleic acid may be assessed prior to administration of a treatment or diagnostic procedure to or on a subject.

This present application is a continuation of co-pending applicationSer. No. 11/733,148, filed Apr. 9, 2007, which claims priority to U.S.Provisional Patent application Ser. No. 60/744,492 filed Apr. 7, 2006,the entire contents of each of which are incorporated herein byreference in its entirety.

The United States Government owns rights in this invention pursuant to agrant from the National Institutes of Health (NIH).

BACKGROUND OF THE INVENTION

I. Field of the Invention

Embodiments of this invention are directed generally to biology andmedicine. In particular the invention is directed to field of genetherapy using AAVP in combination with imaging for providing therapy toa subject.

II. Background

A limitation of many biological-based therapies has been an inability toachieve controlled and effective delivery of biologically activemolecules to tumor cells or their surrounding matrix. The aim ofemploying gene-based therapy is to achieve effective delivery ofbiological products, as a result of gene expression, to their site ofaction within the cell. Gene-based therapy can also provide control overthe level, timing, and duration of action of these biologically activeproducts by including specific promoter/activator elements in thegenetic material transferred resulting in more effective therapeuticintervention. Methods are being developed for controlled gene deliveryto various somatic tissues and tumors using novel formulations of DNA,and for controlling gene expression using cell specific, replicationactivated, and drug-controlled expression systems.

In one approach, gene therapy attempts to target cells in a specificmanner. Thus, a therapeutic gene is linked in some fashion to atargeting molecule in order to deliver the gene into a target cell ortissue. Current methods typically involve linking up a targetingmolecule such as a ligand or antibody that recognizes an internalizingreceptor to either naked DNA or a mammalian cell virus containing thedesired gene. When naked DNA is used it must be condensed in vitro intoa compact geometry for entry into cells. A polycation such as polylysineis commonly used to neutralize the charge on DNA and condense it intotoroid structures. This condensation process, however, is poorlyunderstood and difficult to control, thus, making the manufacturing ofhomogeneous gene therapy drugs extremely challenging.

Bacteriophage (phage), such as lambda and filamentous phage, haveoccasionally been used in efforts to transfer DNA into mammalian cells.In general, transduction of lambda was found to be a relatively rareevent and the expression of the reporter gene was weak. In an effort toenhance transduction efficiency, methods utilizing calcium phosphate orliposomes (which do not specifically target a cell surface receptor)were used in conjunction with lambda. Gene transfer has been observedvia lambda phage using calcium phosphate coprecipitation, or viafilamentous phage using DEAE-dextran or lipopolyamine. However, thesemethods of introducing DNA into mammalian cells are not practical forgene therapy applications, as the transfection efficiency tends to below, non-specific, and transfection is not only cumbersome, but ispromiscuous regarding cell type.

Currently, eukaryotic viruses unquestionably provide superior transgenedelivery and transduction (Kootstra and Verma, 2003; Machida, 2003) butligand-directed targeting of such vectors generally requires ablation oftheir native tropism for mammalian cell membrane receptors (Miller etal., 2003; Mizuguchi and Hayakawa, 2004; White et al., 2004). Incontrast, prokaryotic viruses such as bacteriophage (phage) aregenerally considered poor vehicles for mammalian cell transduction.However, despite their inherent shortcomings as “eukaryotic” viruses,phage particles have no tropism for mammalian cells (Zacher et al.,1980; Barrow and Soothill, 1997; Barbas et al., 2001) and have even beenadapted to transduce such cells (Ivanenkov et al., 1999; Larocca et al.,1999; Poul and Marks, 1999; Piersanti et al., 2004) albeit at lowefficiency.

More reliable means of targeting vectors to specific cells (orreceptors) and of guaranteeing a therapeutically useful degree of genedelivery and expression are thus required, if vectors useful intherapeutic applications are to be achieved.

SUMMARY OF THE INVENTION

Embodiments of the invention are generally directed to compositions andmethods of delivering one or more transgene to a target cell, such as atumor cell, in a site-specific manner to achieve enhanced expression andto constructs and compositions useful in such applications. In certainaspects, expression from a therapeutic nucleic acid may be assessedprior to administration of a treatment or diagnostic procedure to or ona subject. In a further aspect, the determination or evaluation ofexpression in the region or location needed for therapeutic benefit isassessed and any unnecessary or marginal beneficial treatment can bewith held in lieu of alternative treatments.

Without being bound by any particular theory or mechanism, the presentdisclosure is based on the observation that transgene expression may beincreased when the transgene is integrated into a genome with amultiplicity greater than one. Of particular interest is the ability ofcertain chimeric AAVP particles to transduce cells with more than onecopy of the transgene, often as a concatamer. Transduced cells also maybe monitored by the expression of a reporter gene carried by thechimeric AAVP particles. Any transgene may be included in and expressedfrom an AAVP particle of this disclosure.

Certain embodiments of the invention include methods and compositionsfor detecting gene transfer to and/or gene expression in a target tissueof a subject comprising one or more of the following steps:

(a) One step that may be used in the present methods includes deliveringto the target tissue of a subject an AAVP vector containing a reportergene, which may or may not be naturally present in the host subject.Typically, the reporter gene will not be expressed in location or regionto be imaged and/or treated. In certain aspects, the reporter gene is awild-type, a mutant, or a genetically engineered kinase. In a furtheraspect the kinase is a thymidine kinase. In still a further aspect, thekinase is a herpes simplex virus-thymidine kinase gene or humanthymidine kinase type 2. Typically, the transfer vector or AAVP isintroduced to cells of the target tissue, and the reporter gene isexpressed in the cells of the target tissue, thereby generating areporter gene product (protein) which accumulates only in the cellseffectively transfected by the AAVP vector.

(b) Another step that may be used is administering to the host subject alabeled reporter substrate where cells expressing a reporter geneproduct metabolize the labeled reporter substrate to produce a labeledreporter metabolite wherein the labeled reporter substrate comprises aradiolabeled nucleoside analogue.

(c) Yet another step that may be used in the present methods includesnon-invasively imaging a target tissue or cells containing a labeledmetabolite of the reporter substrate. In certain aspects, the subject issubjected to imaging after clearance of residual reporter substrate notmetabolized by the reporter gene product from the host subject therebydetecting gene transfer to and expression in the target tissue. In afurther aspect the subject or subjects tissues are subjected to imagingafter 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more minutes, hours, days, orweeks, depending on the metabolism of clearance of non-metabolizedreporter substrate.

The methods can further comprise waiting for a period of time after step(b) sufficient to allow about, at least, or at most 60, 65, 67, 70, 75,77, 80, 85, 87, 90, 95, 97% or more, including all values and rangesthere between of non-metabolized (reporter substrate not metabolized bythe expression product) by the reporter gene product to clear from thesubject. The non-metabolized substrate may include non-specific labelderived from residual reporter substrate not metabolized. AAVP vectorcan be introduced to the cells of the target tissue by in vitro or invivo transfection (or transduction). In certain aspects, AAVP isadministered intravenously, intratumorally, intrarterially,intrapleurally, intrabronchially, and/or orally.

In certain aspects, a reporter substrate is labeled with a radioisotopesuitable for imaging by positron emission tomography, gamma camera, orsingle-photon emission computed tomography. The reporter substrateand/or metabolite of the reporter substrate are compounds containing astable-isotope nuclide including but not limited to ²H, ¹³C, ¹⁵N and¹⁹F. In a further aspect, the labeled reporter metabolite is imaged bypositron emission tomography. In still further aspects, the labeledreporter metabolite is imaged by gamma camera or single-photon emissioncomputed tomography. In yet still further aspects, the labeled reportersubstrate metabolite is imaged by magnetic resonance imaging.

An AAVP vector may incorporate a reporter gene and suitabletranscription promoter and enhancer elements, ensuring tissue-specific,tissue-selective, or transcription factor-specific, or signaltransduction-specific transcriptional activation of reporter andtherapeutic gene co-expression. In certain aspects, the organ, tissue,cells or a cell is transfected with a reporter gene operably coupled totranscription regulatory elements such as promoter and/or enhancerelements ex vivo (in vitro) prior to administration of the cells or acell to a subject. A labeled 2′-fluoro-nucleoside analogue includes, butis not limited to5-[¹²³I]-2′-fluoro-5-iodo-1β-D-arabinofuranosyl-uracil;5-[¹²⁴I]-2′-fluoro-5-iodo-1β-D-arabinofuranosyl-uracil,5-[¹³¹I]-2′-fluoro-5-fluoro-1β-D-arabinofuranosyl-uracil;2-[¹³¹I]-2′-fluoro-5-methyl-1-β-D-arabinofuranosyl-uracil;5-([¹¹C]-methyl)-2′-fluoro-5-methyl-1-β-D-arabinofuranosyl-uracil;2-[¹¹C]-2′-fluoro-5-ethyl-1-β-D-arabinofuranosyl-uracil;5-([¹¹C]-ethyl)-2′-fluoro-5-ethyl-1-β-D-arabinofuranosyl-uracil;5-(2-[¹⁸F]-ethyl)-2′-fluoro-5-(2-fluoro-ethyl)-1-β-D-arabinofuranosyl-uracil,5-[¹²³I]-2′-fluoro-5-iodovinyl-1-β-D-arabinofuranosyl-uracil;5-[¹²⁴I]-2′-fluoro-5-iodovinyl-1-β-D-arabinofuranosyl-uracil;5-[¹³¹I]-2′-fluoro-5-iodovinyl-1-β-D-arabinofuranosyl-uracil;5-[¹²³I]-2′-fluoro-5-iodo-1-β-D-ribofuranosyl-uracil;5-[¹²⁴I]-2′-fluoro-5-iodo-1-β-D-ribofuranosyl-uracil;5-[¹³¹I]-2′-fluoro-5-iodo-1-β-D-ribofuranosyl-uracil;5-[¹²³I]-2′-fluoro-5-iodovinyl-1-β-D-ribofuranosyl-uracil;5-[¹²⁴I]-2′-fluoro-5-iodovinyl-1-β-D-ribofuranosyl-uracil;5-[¹³¹I]-2′-fluoro-5-iodovinyl-1-β-D-ribofuranosyl-uracil; or9-4-[¹⁸F]fluoro-3-(hydroxymethyl)butyl]guanine.

The imaging data can embody, but is not limited to imaging obtained withmagnetic resonance imaging (MRI), nuclear medicine, positron emissiontomography (PET), computerized tomography (CT), ultrasonography (US),optical imaging, infrared imaging, in vivo microscopy and x-rayradiography. Imaging can be coupled with medical devices, drugs orcompounds, contrast agents or other agents or stimuli that may be usedto elicit additional information from the imaging. Images are obtainedusing these modalities of the lesion, tissue, specimen, system,organism, subject or patient and can be static or dynamic images both intime and/or space.

The imaging can be matched to the tissue, specimen, system, organism, orpatient from which the large scale biological data is obtained. Imaginginformation is extracted from each image, imaging study or studies orexaminations, and can consists of quantitative or qualitative imagingfeatures that may embody but are not limited to differences inmorphology, composition, structure, physiology, gene expression, orfunction of a lesion, a tissue, specimen, system, organism, or patient.Examples of imaging information include but are not limited to imagingfeatures that may be extracted from multi-phase contrast enhanceddynamic CT, functional imaging, magnetic resonance spectroscopy,diffusion tensor imaging, diffusion or perfusion based imaging as wellas targeted imaging encapsulated by nuclear medicine or PET. For anexample see U.S. Patent Publication 20030033616 and 20060223141, whichare incorporated herein by reference in its entirety.

In certain embodiments, the invention includes methods of treating asubject comprising one or more of the following steps:

(a) administering a therapeutic AAVP encoding a reporter to a subjecthaving, suspected of having or at risk of developing a pathologic ordisease condition; and

(b) evaluating in situ expression of the therapeutic AAVP in a tissue orcell targeted for treatment by detecting the encoded reporter orreporter activity.

In certain aspects, the methods can further comprise administering acancer treatment to the subject based on expression of a therapeuticallysufficient level of a therapeutic gene expressed by the AAVP nucleicacid in the target organ, tissue or cell. In a further aspect, secondtherapeutic AAVP can be administered if the expression of the firsttherapeutic AAVP is not expressed at a therapeutically effective level.The second therapeutic AAVP may comprise a second targeting ligand or acombination or ligands. Also, the second AAVP can comprise a secondcontrol element for expression in target organ, tissue, cells, or cell.Evaluation of AAVP expression can be by non-invasive detection of thereporter or an activity of the reporter (e.g., detection of labeledsubtrate metabolized by a reporter protein). In certain aspects, thereporter is a therapeutic protein. In a further aspect, the therapeuticprotein is a prodrug converting enzyme. In still a further aspects, thereporter is an enzyme, and particularly a kinase. In certain embodimentsthe kinase is thymidine kinase, e.g., a HSV-tk or a human tk2.Typically, the kinase modifies or metabolizes a detectably labeledcompound or labeled substrate. In certain aspects the substrate orcompound comprises a detectable label that is detectable byfluorescence, chemiluminescence, surface enhanced raman spectroscopy(SERS), magnetic resonance imaging (MRI), computer tomography (CT), orpositron emission tomography (PET) imaging. In certain aspects, thedetectably labeled compound is a nucleoside analog. The detectably labelcompound may include, but is not limited to fluorodeoxyglucose (FDG);2′-fluoro-2′deoxy-1beta-D-arabionofuranosyl-5-ethyl-uracil (PEAU);5-[¹²³I]-2′-fluoro-5-iodo-1β-D-arabinofuranosyl-uracil;5-[¹²⁴I]-2′-fluoro-5-iodo-1β-D-arabinofuranosyl-uracil;5-[¹³¹I]-2′-fluoro-5-iodo-1β-D-arabinofuranosyl-uracil,5-[¹⁸F]-2′-fluoro-5-fluoro-1-β-D-arabinofuranosyl-uracil; 2-[¹¹I]- and5-([¹¹C]-methyl)-2′-fluoro-5-methyl-1-β-D-arabinofuranosyl-uracil;2-[¹¹C]-2′-fluoro-5-ethyl-1-β-D-arabinofuranosyl-uracil;5-([¹¹C]-ethyl)-2′-fluoro-5-ethyl-1-β-D-arabinofuranosyl-uracil;5-(2-[¹⁸F]-ethyl)-2′-fluoro-5-(2-fluoro-ethyl)-1-β-D-arabinofuranosyl-uracil,5-[¹²³I]-2′-fluoro-5-iodovinyl-1-β-D-arabinofuranosyl-uracil;5-[¹²⁴I]-2′-fluoro-5-iodovinyl-1-β-D-arabinofuranosyl-uracil;5-[¹³¹I]-2′-fluoro-5-iodovinyl-1-β-D-arabinofuranosyl-uracil;5-[¹²³I]-2′-fluoro-5-iodo-1-β-D-ribofuranosyl-uracil;5-[¹²⁴I]-2′-fluoro-5-iodo-1-β-D-ribofuranosyl-uracil;5-[¹³¹I]-2′-fluoro-5-iodo-1-β-D-ribofuranosyl-uracil;5-[¹²³I]-2′-fluoro-5-iodovinyl-1-β-D-ribofuranosyl-uracil;5-[¹²⁴I]-2′-fluoro-5-iodovinyl-1-β-D-ribofuranosyl-uracil;5-[¹³¹I]-2′-fluoro-5-iodovinyl-1-β-D-ribofuranosyl-uracil; or9-4-[¹⁸F]fluoro-3-(hydroxymethyl)butyl]guanine.

In a further aspect the labeled substrate or compound can be labeledwith ¹⁸F, ²⁷⁷Ac, ²¹¹At, ¹²⁸Ba, ¹³¹Ba, ⁷Be, ²⁰⁴Bi, ²⁰⁵Bi, ²⁰⁶Bi, ⁷⁶Br,⁷⁷Br, ⁸²Br, ¹⁰⁹Cd, ⁴⁷Ca, ¹¹C, ¹³C, ¹⁴C, ³⁶Cl, ⁴⁸Cr, ⁵¹Cr, ⁶²Cu, ⁶⁴Cu,⁶⁷Cu, ¹⁶⁵Dy, ¹⁵⁵Eu, ¹⁵³Gd, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁷²Ga, ¹⁹⁸Au, ²H, ³H, ¹⁶⁶Ho,¹¹¹In, ¹¹³In, ¹¹⁵In, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁹Ir, ¹⁹¹Ir, ¹⁹²Ir, ¹⁹⁴Ir, ¹⁹F,⁵²Fe, ⁵⁵Fe, ⁵⁹Fe, ¹⁷⁷Lu, ¹⁵O, ¹⁹¹Os, ¹⁰⁹Pd, ³²P, ³³P, ⁴²K, ²²⁶Ra, ¹⁸⁶Re,¹⁸⁸Re, ⁸²Rb, ¹⁵³Sm, ⁴⁶Sc, ⁴⁷Sc, ⁷²Se, ⁷⁵Se, ¹⁰⁵Ag, ¹⁵N, ²²Na, ²⁴Na,⁸⁹Sr, ³⁵S, ³⁸S, ¹⁷⁷Ta, ⁹⁶Tc, ^(99m)Tc, ²⁰¹Tl, ²⁰²Tl, ¹¹³Sn, ^(117m)Sn,¹²¹Sn, ¹¹⁶Yb, ¹⁶⁹Yb, ¹⁷⁵Yb, ⁸⁸Y, ⁹⁰Y, ⁶²Zn, or ⁶⁵Zn. In particularaspects the detectable label is ¹³¹I, ¹²⁵I, ¹²³I, ¹¹¹I, ^(99m)Tc, ⁹⁰Y,¹⁸⁶Re, ¹⁸⁸Re, ³²P, ¹⁵³Sm, ⁶⁷Ga, ²⁰¹Tl, ⁷⁷Br, or ¹⁸F label.

An AAVP of the invention may comprise a moiety that selectively targetsa tissue or cell targeted for treatment. In certain aspects, the moietyis encoded by or coupled to a capsid protein and/or a recombinant capsidprotein of an AAVP. In certain aspects, a capsid protein comprises atargeting peptide. A targeting peptide can be a cyclic peptide, abicyclic, and/or a linear peptide. The targeting peptide selectivelybinds a cell expressing an integrin on the cell surface. An integrin canbe a αvβ3 or αvβ5 integrin. In a further aspect, peptide comprises anRGD motif. In still a further aspect, the peptide can selectively bindsa cell expressing a transferrin receptor, such a peptide can include anamino acid sequence comprising CRTIGPSVC.

In certain aspects, a subject can have, is suspected of having, or atrisk of developing a hyperproliferative disease. Hyperproliferativedisease include, but are not limited to fibrosarcoma, myosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer,pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer,cancer of the head and neck, skin cancer, brain cancer, squamous cellcarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcancer, testicular cancer, small cell lung carcinoma, non-small celllung carcinoma, bladder carcinoma, epithelial carcinoma, glioma,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposisarcoma. In a particular aspect the hyperproliferative disease isglioma.

In a further embodiment, a reporter and/or therapeutic gene isoperatively coupled to a tissue or cell selective promoter, or a tissueor cell specific promoter. In certain aspects, evaluating expressioncomprises administering a labeled compound or substrate that ismetabolized by a cell expressing the AAVP nucleic acid and typically notmetabolized to a significant extend by non-target tissues.

A therapeutic AAVP may also encode a second therapeutic gene. The secondtherapeutic gene can be, but is not limited to a tumor suppressor, aninhibitory RNA, an inhibitory DNA, or a prodrug converting enzyme.

In still further embodiments, compositions of the invention can includea therapeutic AAVP nucleic acid comprising a nucleic acid segmentcomprising an inhibitory RNA or inhibitory DNA. The inhibitory RNA canbe a siRNA, a miRNA, or an antisense RNA or DNA. In certain aspects anAAVP nucleic acid is comprised in a phage particle. In a further aspectthe particle comprises a targeting ligand as described herein.

Certain embodiments include compositions and methods for modulating theexpression of a gene comprising administering an AAVP nucleic acid orparticle comprising such.

In accordance with the present invention, a selected gene or polypeptidemay refer to any protein, polypeptide, or peptide. A therapeutic gene orpolypeptide is a gene or polypeptide which can be administered to asubject for the purpose of treating or preventing a disease. Forexample, a therapeutic gene can be a gene administered to a subject fortreatment or prevention of cancer. Examples of therapeutic genesinclude, but are not limited to, Rb, CFTR, p16, p21, p27, p57, p73,C-CAM, APC, CTS-1, zac1, scFV ras, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II,BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF, G-CSF, thymidine kinase,Bax, Bak, Bik, Bim, Bid, Bad, Harakiri, Fas-L, mda-7, fus, interferon α,interferon β, interferon γ, p53, ABLI, BLC1, BLC6, CBFA1, CBL, CSFIR,ERBA, ERBB, EBRB2, ETS1, ETS2, ETV6, FGR, FOX, FYN, HCR, HRAS, JUN,KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET,SRC, TAL1, TCL3, YES, MADH4, RB1, TP53, WT1, TNF, BDNF, CNTF, NGF, IGF,GMF, aFGF, bFGF, NT3, NT5, ApoAI, ApoAIV, ApoE, Rap1A, cytosinedeaminase, Fab, ScFv, BRCA2, zac1, ATM, HIC-1, DPC-4, FHIT, PTEN, ING1,NOEY1, NOEY2, OVCA1, MADR2, 53BP2, IRF-1, zac1, DBCCR-1, rks-3, COX-1,TFPI, PGS, Dp, E2F, ras, myc, neu, raf, erb, fms, trk, ret, gsp, hst,abl, E1A, p300, VEGF, FGF, thrombospondin, BAI-1, GDAIF, or MCC.

Other examples of therapeutic genes include genes encoding enzymes.Examples include, but are not limited to, ACP desaturase, an ACPhydroxylase, an ADP-glucose pyrophorylase, an ATPase, an alcoholdehydrogenase, an amylase, an amyloglucosidase, a catalase, a cellulase,a cyclooxygenase, a decarboxylase, a dextrinase, an esterase, a DNApolymerase, an RNA polymerase, a hyaluron synthase, a galactosidase, aglucanase, a glucose oxidase, a GTPase, a helicase, a hemicellulase, ahyaluronidase, an integrase, an invertase, an isomerase, a kinase, alactase, a lipase, a lipoxygenase, a lyase, a lysozyme, apectinesterase, a peroxidase, a phosphatase, a phospholipase, aphosphorylase, a polygalacturonase, a proteinase, a peptidease, apullanase, a recombinase, a reverse transcriptase, a topoisomerase, axylanase, a reporter gene, an interleukin, or a cytokine.

Further examples of therapeutic genes include the gene encodingcarbamoyl synthetase I, ornithine transcarbamylase, arginosuccinatesynthetase, arginosuccinate lyase, arginase, fumarylacetoacetatehydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin,glucose-6-phosphatase, low-density-lipoprotein receptor, porphobilinogendeaminase, factor VIII, factor IX, cystathione beta.-synthase, branchedchain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase,propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoAdehydrogenase, insulin, -glucosidase, pyruvate carboxylase, hepaticphosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein,T-protein, Menkes disease copper-transporting ATPase, Wilson's diseasecopper-transporting ATPase, cytosine deaminase, hypoxanthine-guaninephosphoribosyltransferase, galactose-1-phosphate uridyltransferase,phenylalanine hydroxylase, glucocerbrosidase, sphingomyelinase,-L-iduronidase, glucose-6-phosphate dehydrogenase, HSV thymidine kinase,or human thymidine kinase.

Therapeutic genes also include genes encoding hormones. Examplesinclude, but are not limited to, genes encoding growth hormone,prolactin, placental lactogen, luteinizing hormone, follicle-stimulatinghormone, chorionic gonadotropin, thyroid-stimulating hormone, leptin,adrenocorticotropin, angiotensin I, angiotensin II, β-endorphin,β-melanocyte stimulating hormone, cholecystokinin, endothelin I,galanin, gastric inhibitory peptide, glucagon, insulin, lipotropins,neurophysins, somatostatin, calcitonin, calcitonin gene related peptide,β-calcitonin gene related peptide, hypercalcemia of malignancy factor,parathyroid hormone-related protein, parathyroid hormone-relatedprotein, glucagon-like peptide, pancreastatin, pancreatic peptide,peptide YY, PHM, secretin, vasoactive intestinal peptide, oxytocin,vasopressin, vasotocin, enkephalinamide, metorphinamide, alphamelanocyte stimulating hormone, atrial natriuretic factor, amylin,amyloid P component, corticotropin releasing hormone, growth hormonereleasing factor, luteinizing hormone-releasing hormone, neuropeptide Y,substance K, substance P, or thyrotropin releasing hormone.

Other embodiments of the invention are discussed throughout thisapplication. Any embodiment discussed with respect to one aspect of theinvention applies to other aspects of the invention as well and viceversa. The embodiments in the Example section are understood to beembodiments of the invention that are applicable to all aspects of theinvention.

The terms “inhibiting,” “reducing,” or “prevention,” or any variation ofthese terms, when used in the claims and/or the specification includesany measurable decrease or complete inhibition to achieve a desiredresult.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

It is contemplated that any embodiment discussed herein can beimplemented with respect to any method or composition of the invention,and vice versa. Furthermore, compositions and kits of the invention canbe used to achieve methods of the invention.

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein. Some specific example embodimentsof the disclosure may be understood by referring, in part, to thefollowing description and the accompanying drawings.

FIGS. 1A-1E FIG. 1A is a graph showing binding of RGD-4C AAVP tomammalian cells expressing αν integrins, in contrast to the non-targetedAAVP or AAVP displaying negative control peptides such as RGE-4C orvarious scrambled versions of the RGD-4C sequence. FIG. 1B is an imageshowing RGD-4C AAVP carrying reporter genes indicating ligand-directedinternalization. FIG. 1C shows targeted gene transfer mediated by RGD-4CAAVP-β-galactosidase to KS 1767 cells. FIG. 1D shows inhibition oftransduction by the synthetic RGD-4C peptide, but not by an unrelatedcontrol peptide; nonspecific transduction levels were determined byusing non-targeted AAVP. An anti-β-gal antibody was used for stainingand gene expression was detected by immunofluorescence. FIG. 1E showsthe rescue of recombinant AAV from cells infected with RGD-4C AAVP.Human 293 cells were incubated with targeted RGD-4C AAVP-GFP (10⁶transducing units/cell) or negative controls (targeted non-chimeraRGD-4C phage-GFP, nontargeted AAVP-GFP). Four days after infection,cells were transfected with an AAV rep- and cap-expressing plasmid andsuperinfected with wild-type adenovirus type 5 (Ad). Cells wereharvested 72 h post-adenoviral infection and supernatants were then usedto infect new 293 cells. GFP expression was analyzed by flow cytometry48 h later. Mean increases in recombinant AAV-GFP produced overbackground after rescue from each construct are shown.

FIGS. 2A-2C FIG. 2A is a histogram showing a summary of flow cytometricanalyses of 293 clones stably transduced with either AAVP-GFPneo (n=9)or phage-GFPneo (n=9). Open triangles indicate percentages of greenfluorescent protein (GFP)-positive cells and black bars represent GFPexpression levels (mean fluorescent intensity; MFI). FIG. 2B showsSouthern blot analysis of the persistence of transgene cassette inclonal cell lines transduced with RGD-4C AAVP-GFPneo or RGD-4Cphage-GFPneo. Total cellular DNA from non-transduced 293 parental cellsor each of the transduced cell clones (#1-9 for each group) wasdouble-digested with AflII-XhoI (one restriction digestion site per eachenzyme within the construct DNA flanking the transgene cassette). FIG.2C shows analysis of potential head-to-tail concatemers of the transgenecassette by Southern blot. Total cellular DNA was digested with Xho-I(single restriction digestion site within the transgene cassette next tothe 3′ ITR) prior to Southern blotting.

FIGS. 3A-3B FIG. 3A shows immunohistochemical staining against phage inKS 1767-derived xenografts after systemic administration (intravenouslythrough the tail vein) of RGD-4C AAVP (5×10¹⁰ TU) or negative controls(non-targeted AAVP, scrambled RGD-4C AAVP, or RGE-4C AAVP) into deeplyanesthetized nude mice bearing KS1767-derived tumor xenografts. AAVPconstructs were allowed to circulate for 5 min, followed by perfusionand surgical removal of tumors. A polyclonal antibody against phage wasused for staining on paraffin-embedded tumor sections. Arrows point tophage staining in tumor blood vessels. FIG. 3B shows immunofluorescenceanalysis of GFP expression in KS1767-derived xenografts at day 7 aftersystemic administration of either RGD 4C AAVP-GFP or negative controls(non-targeted, scrambled or mutant) as indicated.

FIGS. 4A-4E FIG. 4A shows in vivo bioluminescent imaging of fireflyluciferase expression after systemic delivery of targeted AAVP. Nudemice bearing DU145-derived tumor xenografts received a systemicsingle-dose of either RGD-4C AAVP-Luc (5×10¹¹ TU, intravenous) orcontrols (non-targeted AAVP-Luc, or scrambled RGD-4C AAVP-Luc). Ten dayslater, bioluminescence imaging (BLI) of tumor-bearing mice was performedto assess the transgene expression. Calibration scales are provided inpanels. FIG. 4B shows multi-tracer PET imaging in tumor-bearing miceafter systemic delivery of targeted RGD-4C AAVP-HSVtk. Nude mice bearingDU145-derived tumor xenografts (n=9 tumor-bearing mice per each cohort)received a systemic single-dose (5×10¹¹ TU, intravenous) RGD-4CAAVP-HSVtk or non-targeted AAVP-HSVtk. PET images with [¹⁸F]-FDG and[¹⁸F]-FEAU obtained before and after GCV treatment are presented. T,tumor; H, heart; BR, brain; BL, bladder. Calibration scales are providedin panels. Overimposition of PET images and photographic images ofrepresentative tumor-bearing mice was performed to simplify theinterpretation of [¹⁸F]-FDG and [¹⁸F]-FEAU biodistribution. FIG. 4Cshows growth curves of individual tumor-xenografts after AAVPadministration. FIG. 4D shows temporal dynamics of HSVtk gene expressionas assessed by repetitive PET imaging with [¹⁸F]-FEAU at different dayspost AAVP administration. FIG. 4E shows changes in tumor viabilitybefore and after GCV therapy as assessed with [¹⁸F]-FDG PET.

FIGS. 5A-5G FIG. 5A is a graph showing the plotted mean tumorvolumes±standard deviations (SD) over time. Cohorts of immunodeficientnude mice with established human xenografts (size-matched atapproximately 50 mm²) derived from KS 1767 cells were used. The micereceived a single systemic administration (5×10¹⁰ TU, intravenous) ofeither RGD-4C AAVP-HSVtk or controls (non-targeted AAVP-HSVtk, RGD-4CAAVP-GFP, or vehicle alone). Gancyclovir (GCV) was administered to micefrom post-treatment day 2 until the end of the experiments. All micereceived GCV except for an additional control group treated with RGD-4CAAVP-HSVtk but without GCV afterwards. FIG. 5B is a graph showing theplotted mean tumor volumes±standard deviations (SD) over time. Cohortsof immunodeficient nude mice with established human xenografts(size-matched at approximately 50 mm²) derived from bladder UC3carcinoma cells. The mice received a single systemic administration(5×10¹⁰ TU, intravenous) of either RGD-4C AAVP-HSVtk or controls(non-targeted AAVP-HSVtk, RGD-4C AAVP-GFP, or vehicle alone).Gancyclovir (GCV) was administered to mice from post-treatment day 2until the end of the experiments. All mice received GCV except for anadditional control group treated with RGD-4C AAVP-HSVtk but without GCVafterwards. FIG. 5C is a graph showing the plotted mean tumorvolumes±standard deviations (SD) over time. Cohorts of immunodeficientnude mice with established human xenografts (size-matched atapproximately 50 mm²) derived from prostate DU145 carcinoma cells. Themice received a single systemic administration (5×10¹⁰ TU, intravenous)of either RGD-4C AAVP-HSVtk or controls (non-targeted AAVP-HSVtk, RGD-4CAAVP-GFP, or vehicle alone). Gancyclovir (GCV) was administered to micefrom post-treatment day 2 until the end of the experiments. All micereceived GCV except for an additional control group treated with RGD-4CAAVP-HSVtk but without GCV afterwards. Shown are the plotted mean tumorvolumes±standard deviations (SD) over time. FIG. 5D is a graph showinggrowth inhibition of large DU145-derived xenografts (at approximately150 mm²) by a single systemic dose (5×10¹⁰ TU, intravenous) of RGD-4CAAVP-HSVtk. FIG. 5E is a graph showing inhibition of tumor growth ofEF43-FGF4 mouse mammary carcinoma (size-matched at approximately 50 mm²)in immunocompetent BALB/c mice by a single intravenous dose (5×10¹⁰ TU)of RGD-4C AAVP-HSVtk. FIG. 5F is a graph showing long-term efficiency ofRGD-4C AAVP-HSVtk and GCV by repeated doses of AAVP (5×10¹⁰ TU each,intravenous) to immunocompetent BALB/c mice bearing isogenic EF43-FGF4tumors. Therapy results were consistently observed in independentexperiments with tumor-bearing mice cohorts (n=10 mice per treatmentgroup). Arrows indicate times of AAVP administration. FIG. 5G is a graphshowing the effect of the humoral immune response against phage ontherapy with RGD-4C AAVP-HSVtk. BALB/c immunocompetent mice (n=7 miceper group) were first “vaccinated” by receiving three systemic doses ofRGD-4C AAVP-GFP (10¹⁰ TU per week for three weeks, intravenous). Micewere then implanted with EF43-FGF4 cells. When tumors were established(size-matched at approximately 50 mm²), tumor-bearing mice received onesystemic single-dose of RGD-4C AAVP-HSVtk or non-targeted AAVP-HSVtkfollowed by GCV maintenance (started at day 2 post administration of theAAVP). Serum samples were collected from mice pre- and post-vaccinationwas started and again at the end of vaccination scheme before AAVPadministration in order to confirm the presence of high titers (up toapproximately 1:10,000) of circulating anti-phage IgG by ELISA.Vaccination did not appear to affect the anti-tumor effects, despite theanti-phage antibodies presence.

FIGS. 6A-6B FIG. 6A is an image of tumor-bearing mice (upper panel) andcorresponding surgically removed tumors (lower panel) from all theexperimental groups of therapy (EF43-FGF4 mammary tumors in BALB/cimmunocompetent mice). FIG. 6B is histopathologic analysis of EF43-FGF4treated tumors. EF43-FGF4 tumors were recovered, sectioned, and stained.Non-targeted AAVP-HSVtk-treated tumors (left panels), the border betweenthe outer rims and central tumor areas (middle panels), and centraltumor areas of RGD-4C AAVP-HSVtk-treated tumors (right panel) are shownas high-magnification views from the low-magnification inserts of serialtumor sections. Hematoxylin and eosin (H&E) staining, anti-CD31immunostaining and TUNEL staining of tumor sections are shown. Arrowspoint to tumor blood vessels and apoptotic cells.

FIG. 7 FIG. 7 is a schematic showing the formation the construction ofRGD-4C AAVP, cloning strategy and structure of the resulting vectors aredepicted.

FIGS. 8A-8C FIG. 8A shows a Southern blot analysis of the DNA fromRGD-4C AAVP-GFPneo or RGD-4C phage-GFPneo in 293 transduced clonal celllines. Total cellular DNA from non-transduced 293 parental cells orindividual stable cell clones transduced (#1-9 for each vector) wasincubated with StuI (no restriction digest site within the vector DNA),Xba-I (single restriction digest site within the vector DNA), orSacI-MluI. Resulting DNA fragments were separated on 0.8% agarose gel,transferred to a nylon membrane and hybridized with a labeled neo probeas indicated. Digested vector plasmids were also used as controls. FIG.8B shows a PCR analysis of concatemers of the transgene cassette in 293clonal cell lines stably transduced with RGD-4C phage-GFPneo or RGD-4CAAVPGFPneo. Non-transduced 293 parental cells served as a negativecontrol; additional negative controls and a 100-bp molecular marker(Invitrogen) are also shown as indicated. Nested PCR with primersannealing close to the 5′ and 3′ end of the transgene cassette wasperformed to identify concatemeric forms in the DNAs corresponding tothe RGD-4C AAVP and RGD-4C phage. Arrows indicate primers (H, head; T,tail). PCR-amplification products were detected in 2% agarose gels andrevealed concatemeric vector DNA exclusively in the cell clonestransduced with AAVP. FIG. 8C shows sequencing results of differentconcatemeric forms in AAVP DNA (capital letters denote 3′ end oftransgene cassette, italic letters denote 5′ end of transgene cassette),revealed Head-to-Tail concatemers with deleted ITRs.

FIG. 9 FIG. 9 is an image showing immunostaining of the reporter geneexpression GFP in control organs brain, liver, pancreas, and kidney atday 7 after an intravenous dose (5×10¹⁰ TU) of either RGD-4C AAVP-GFP ornon-targeted AAVP-GFP into mice.

FIGS. 10A-10B FIG. 10A is an image of immunohistochemical stainingagainst AAVP after intravenous administration of RGD-4C AAVP (rightpanel) or non-targeted AAVP (middle panel) into immunocompetent BALB/cmice bearing EF43-FGF4 tumors. Constructs were allowed to circulate for5 min, followed by perfusion and tissue recovery as described. Apolyclonal antibody against phage was used for staining Left panel showstumor blood vessel staining by using an anti-CD31 antibody. Arrows pointto tumor blood vessels. FIG. 10B is an image of immunofluorescenceanalysis of GFP expression in EF43-FGF4 tumors at 1 week afterintravenous administration of non-targeted AAVP-GFP (left panel) orRGD-4C AAVP-GFP (middle panel) into mice bearing EF43-FGF4 tumors.Immunostaining against αv-integrin in EF43-FGF4 tumors is also shown(right panel).

FIG. 11 FIG. 11 shows images of histological analysis of control organsliver, heart, and kidney from mice treated with non-targeted AAVP-HSVtk,RGD-4C AAVP-HSVtk vectors, or vehicle alone plus GCV maintenance areshown. No histological signs of toxicity were detected in these organsby H&E staining.

FIGS. 12A-12C Mammalian cells infected with RGD-targeted AAVP expressesa functional gene product. (FIG. 12A) Human melanoma cells, M21 wereinfected with AAVP; non-targeted TNF-α expressing phage fdTNF-α (upperpanel) and targeted TNF-α expressing virus RGDTNF-α (lower panel) anddetected using fd specific primary antibody followed by FITC labeledsecondary antibody. (FIG. 12B) M21 cells infected with PBS, emptynon-targeted virus (fd), empty targeted virus (RGD), non-targeted TNF-αexpressing virus (fdTNF-α) and targeted TNF-α expressing virus RGDTNF-α.5 days after infection culture supernatant was analyzed to measure TNF-αby ELISA. The culture supernatant 12 days after the infection is alsoshown. (FIG. 12C) Human umbilical vein endothelial cells (HUVEC) weretreated with day 5 supernatant from M21 cells infected various groups;PBS, fd (non-targeted null virus), RGD (targeted null virus), fdTNF(non-targeted TNF-α expressing virus) and RGDTNF (targeted TNF-αexpressing virus) and analyzed for tissue factor (TF) production.Recombinant TNF-α was used as a positive control. To check thespecificity, M21 supernatant from RGDTNF infected cells incubated withTNF-α specific antibody, before applying onto HUVEC. Day 23 supernatantafter infection also been tested for TF secretion

FIGS. 13A-13H AAVP is specifically targeted to tumor vasculature. Humanmelanoma xenografts injected with either PBS or RGDTNF-α stained withbacteriophage specific antibody and CD31 blood vessel antibody. Thedetection was done using Alexa Flour 488, Alexa Flour 594 and DAPI tovisualize blood vessels, AAVP and cell nuclei respectively (250×). Noneof the animals injected with PBS showed presence of AAVP at any timepoint. A representative tumor section from animal injected with PBS for15 min is shown (FIG. 13A). The animals injected with AAVP expressingRGDTNF-α, showed colocalization of virus particles in the blood vesselsas early as 15 min (FIG. 13B) and in subsequent time points: day 1 (FIG.13C), day 2 (FIG. 13D), day 3 (FIG. 13E), day 4 (FIG. 13F), day 8 (FIG.13G), and day 10 (FIG. 13H).

FIGS. 14A-14E AAVP particles were not detected in normal liver tissue.The liver from animals with human melanoma xenografts injected withRGDTNF-α stained with bacteriophage specific antibody and CD31 bloodvessel antibody. The detection was done using Alexa Flour 488, AlexaFlour 594 and DAPI to visualize blood vessels, AAVP and cell nucleirespectively (250×). The animals injected with AAVP expressing RGDTNF-α,showed some staining of virus particles at day 1 (FIG. 14A) in the livertissue. However, the virus staining was reduced to minimal levels by day2 (FIG. 14B) with no virus staining observed at day 3 (FIG. 14C), day 8(FIG. 14D) and day 10 (FIG. 14E) time points. All the liver tissues atdifferent time points showed good vessels staining.

FIGS. 15A-15E AAVP particles were not detected in normal kidney tissue.The kidney from animals with human melanoma xenografts injected withRGDTNF-α was stained with the bacteriophage specific antibody and CD31blood vessel antibody. The detection was done using Alexa Flour 488,Alexa Flour 594 and DAPI to visualize blood vessels, AAVP and cellnuclei respectively (250×). We did not observe presence of AAVP inkidney in any of the time points tested, day 1 (FIG. 15A), day 2 (FIG.15B), day 3 (FIG. 15C), day 8 (FIG. 15D) and day 10 (FIG. 15E).Nevertheless, all the kidney tissues at different time points showedgood vessels staining.

FIG. 16 In vivo, AAVP expresses specific gene product. Frozen sectionsfrom animals with human melanoma xenografts injected with either PBS orRGDTNF-α were used to extract total protein. 50 μgs of total protein wasused to measure TNF-α protein levels by ELISA in duplicates. Basallevels of endogenous TNF-α expression were observed in all the tissuestested. Animals injected with PBS did not show any increase over theendogenous levels at any time points tested. Mice injected with RGDTNF-αAAVP showed TNF-α expression starting at day 4 and gradually increasingup to day 10.

FIGS. 17A-17B AAVP expresses TNF-α in the vasculature and inducesapoptosis in vivo. (FIG. 17A) Frozen sections from animals with humanmelanoma xenografts injected with RGDTNF-α AAVP stained with TNF-αspecific antibody using immunohistochemical analysis. TNF-α staining isseen as a brown color stain around the blood vessels (left panel, 100×;right panel 400×). (FIG. 17B) Frozen sections from animals with humanmelanoma xenografts injected with RGDTNF-α AAVP stained to detectapoptotic cells. The blood vessel and surrounding tumor cells showedapoptosis seen in blue colored cells stained with TACS blue label (leftpanel, 200×). The samples are counterstained with nuclear fast red. Theright panel shows blood vessels stained with CD31 specific antibody(200×).

FIGS. 18A-18B Treatment of TNF-α sensitive human melanoma, M21 (FIG.18A) and TNF-α resistant human melanoma Pmel (FIG. 18B) with AAVP. Nudemice with subcutaneously implanted M21/Pmel tumors were treated withAAVP systemically through tail vein injection and tumor volumes weremeasured at different time points. The tumor volumes plotted against thedifferent days post treatment. (FIG. 18A) The animals treated withRGDTNF-α showed statistically significant (p<0.05) reduction in tumorvolume starting at day 20. (FIG. 18B) The TNF-α resistant human melanomawas made sensitive to TNF-α therapy by delivery of EMAP-II through AAVPfollowed by treatment with recombinant TNF-α. Mice treated with eitherrTNF-α or targeted AAVP expressing EMAP-II (RGDEMAP-II) alone showedvery little effect and were not significantly different than PBS orfdEMAP-II group. Mice treated with combination of RGD-EMAP-II virus andrTNF-α showed significant reduction in tumor volume (p=0.007).

FIGS. 19A-19B Transduction of human glioma cells in culture by CRTIGPSVC(SEQ ID NO:1) AAVP—Luc U87 human-derived glioma cells were seeded onto24 wells plate at the concentration of 40,000 cells/well and culturedO.N. at 37° C. Next day, cells were incubated with AAVP, according withNature Method's protocol. RGD-4C AAVP GFP and RGD-4C AAVP Luc wer usedas positive control for transduction efficiency. The images were takenat day 7. Phage uptake is low but increases dramatically when cells arecultured in Iron AAVP hydrogel (last column of the plate and graphic).

While the present disclosure is susceptible to various modifications andalternative forms, specific example embodiments have been shown in thefigures and are herein described in more detail. It should beunderstood, however, that the description of specific exampleembodiments is not intended to limit the invention to the particularforms disclosed, but on the contrary, this disclosure is to cover allmodifications and equivalents as illustrated, in part, by the appendedclaims.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure, according to certain embodiments, is generallydirected to methods of delivering one or more transgenes to a targetcell, such as a tumor cell, in a site-specific manner to achieveenhanced expression and to constructs and compositions useful in suchapplications. In certain aspects, expression from a therapeutic nucleicacid may be assessed prior to administration of a treatment ordiagnostic procedure to or on a subject. In a further aspect, thedetermination or evaluation of expression in the region or locationneeded for therapeutic benefit is assessed and any unnecessary ormarginal beneficial treatment can be with held in lieu of alternativetreatments.

Without wishing to be bound by theory or mechanism, the presentdisclosure is based on the observation that transgene expression may beincreased when the transgene is integrated into a genome with amultiplicity greater than one. Of particular interest is the ability ofcertain chimeric AAVP particles to transduce cells with more than onecopy of the transgene, often as a concatamer. Transduced cells also maybe monitored by the expression of a reporter gene carried by thechimeric AAVP particles. Any transgene may be included in and expressedfrom an AAVP particle of this disclosure.

I. ADENO-ASSOCIATED VIRAL/PHAGE (AAVP) PARTICLE

Adeno-associated virus (AAV) is a defective member of the parvovirusfamily. The AAV genome is encapsulated as a single-stranded DNA moleculeof plus or minus polarity. Strands of both polarities are packaged, butin separate virus particles and both strands are infectious. Thesingle-stranded DNA genome of the human adeno-associated virus type 2(AAV2) is 4681 base pairs in length and is flanked by inverted terminalrepeat sequences (ITRs) of 145 base pairs each. In addition, the viralrep protein appears to mediate nonhomologous recombination through theITRs. Accordingly, as parvoviral genomes have ITRs at each end whichplay a role in recombination and which are generally required forparvoviral replication and packaging, AAVPs of the present disclosuregenerally contain all or a portion of at least one of the ITRs or afunctional equivalent thereof.

AAVs may be readily obtained and their use as vectors for gene deliveryhas been described in, for example, Muzyczka, 1992; U.S. Pat. No.4,797,368, and PCT publication WO 91/18088. Construction of AAV vectorsis described in a number of publications, including Lebkowski et al.,1988; Tratschin et al., 1985; Hermonat and Muzyczka, 1984.

The present disclosure provides adeno-associated viral (AAV)bacteriophage vectors (such as AAV-M13 vectors) (AAVPs) that areproduced in bacteria and methods for expressing a transgene in a targetcell, such as a tumor cell, by transducing the cell with the AAVP. Oncepurified, a targeted bacteriophage particle containing the bacteriophageand AAV sequences with transgene cassette are used to transfectmammalian cells. Following internalization of the vector within themammalian cell, the transgene is integrated into the genome of thetarget cells. The term “vector” as used herein is defined as a nucleicacid vehicle for the delivery of a nucleic acid of interest into a cell.The vector may be a linear molecule or a circular molecule.

An AAVP combines selected elements of both phage and AAV vector systems,providing a vector that is simple to produce in bacteria with nopackaging limit, while allowing infection of mammalian cells combinedwith integration into the host chromosome. Vectors containing many ofthe appropriate elements are commercially available, and can be furthermodified by standard methodologies to include the necessary sequences.At minimum, for use with the methods of the present disclosure, thevector must accept a cassette containing a promoter and a transgene. TheAAVP vectors of the present disclosure allow for enhanced transgeneexpression upon incorporation into the target cell genome. In certainembodiments, the transgene may be integrated into the genome of thetarget cell as a concatamer.

Among other things, AAVPs do not require helper viruses or trans-actingfactors. In addition, the native tropism of AAV for mammalian cells iseliminated since there is not AAV capsid formation.

A. AAVP Targeting

The AAVPs of the present disclosure can be targeted to specificreceptors by the expression of ligands on the surface of the phageparticle. In certain embodiments, peptides or other moieties that allowor promote the escape of the vectors (and any molecule attached theretoor enclosed therein) from the endosome may be incorporated and expressedon the surface of the phage. Such “other moieties” include moleculesthat are not themselves peptides but which have the ability to disruptthe endosomal membrane, thereby facilitating the escape of the vector,and molecules that otherwise mimic the endosomal escape properties ofthe within described peptide sequences (see, e.g., published PCTPublication WO 96/10038 and Wagner et al., 1992).

The AAVP of the present disclosure are generally comprised offilamentous phage particles expressing one or more preselected ligandson the particle surface, irrespective of the manner in which the ligandsare attached. Therefore, whether the means of attachment for a ligand iscovalent or via a capsid protein, the AAVP of the present disclosure areable to deliver one or more transgenes to target cells by ligand bindingto a receptor followed by internalization of the vectors. For example,the ligand particle expressed on the particle surface may be bicyclicCDCRGDCFC (RGD-4C) (SEQ ID NO:2) peptide that selectively binds αvβ3 andαvβ5 integrins. These integrins are highly overexpressed on invadingtumor endothelial cells. As used herein, “filamentous phage particle”refers to particles containing either a phage genome or a phagemidgenome. The particles may contain other molecules in addition tofilamentous capsid proteins. As used herein, “ligand” refers to anypeptide, polypeptide, protein or non-protein, such as a peptidomimetic,that is capable of binding to a cell-surface molecule and internalizing.As used herein, to “binding to a receptor” refers to the ability of aligand to specifically recognize and detectably bind to a receptor, asassayed by standard in vitro or in vivo assays.

Typically, the AAVPs of the present disclosure include anoligonucleotide insert in the phage plasmid genome encoding a targetingpeptide, which allows for ligand-receptor targeting properties of thevectors.

Phage capsid proteins or capsid proteins may be modified by coupling orfusing all or part of a capsid protein polynucleotide or protein encodedby the polynucleotide to a targeting ligand. The targeting ligand maydirect, redirect, target or enhance binding of the AAVP of the inventionto a specific cell, tissue and/or organ.

Targeted viruses were originally created to overcome problemsencountered by gene therapy vectors' natural host cell tropisms. Inrecent years, many gene therapy patents have issued wherein the vectorcontains a heterologous polypeptide used to target the vector tospecific cells, such as vectors containing chimeric fusion glycoproteins(Kayman et al., U.S. Pat. No. 5,643,756, incorporated herein byreference) and vectors that contain an antibody to a virus capsidprotein (Cotten et al., U.S. Pat. No. 5,693,509). An AAVP of theinvention may be genetically modified in such a way that the particle istargeted to a particular cell type (e.g., smooth muscle cells, hepaticcells, renal cells, fibroblasts, keratinocytes, stem cells, mesenchymalstem cells, bone marrow cells, chondrocyte, epithelial cells, intestinalcells, neoplastic or cancerous cells and others known in the art) suchthat the nucleic acid genome is delivered to a target non-dividing, atarget dividing cell, or a target cell that has a proliferative or otherdisorder. One way of targeting viruses is to direct the virus to atarget cell by preferentially binding to cells having a molecule on theexternal surface of the cell. This method of targeting the virusutilizes expression or incorporation of a targeting ligand on or intothe capsid of the virus to assist in targeting the virus to cells ortissues that have a receptor or binding molecule which interacts withthe targeting ligand on the surface of the virus. After infection of acell by the virus the genetic material can be processed and expressed inthe host cell. The genetic material may be integrated into the genome ofthe host cell or episomally maintained within the host cell.

In certain embodiments of the invention, a capsid protein may bemodified to include a targeting moiety such that an AAVP may bedelivered to specific cell types or tissues. The targeting specificityof the ligand-based delivery systems are based on the distribution ofthe ligand receptors on different cell types. A targeting ligand mayeither be non-covalently or covalently associated with a capsid protein.

In certain embodiments, a heterologous nucleic acid sequence of interestmay be inserted into the viral vector of the invention. For example, acapsid protein may be operatively coupled to a ligand for a receptor ona specific target cell.

Targeting ligands are any ligand specific for a characteristic componentof the targeted region. Preferred targeting ligands include proteinssuch as polyclonal or monoclonal antibodies, antibody fragments, orchimeric antibodies, enzymes, peptides or hormones, or sugars such asmono-, oligo- and polysaccharides. In certain embodiments of theinvention, contemplated targeting ligands interact with integrins,proteoglycans, glycoproteins, receptors, or transporters. Suitableligands include any that are specific or selective for cells of thetarget organ, or for structures of the target organ exposed to thecirculation as a result of local pathology, such as tumors.

In certain embodiments of the present invention, in order to enhance thetransduction of resistant cells, to increase transduction of targetcells, or to limit transduction of undesired cells, antibody or cyclicpeptide targeting moieties (ligands) may be associated with the AAVP.The antibody targeting moiety in particular example is a monoclonalanti-EGF receptor antibody. EGF receptors are distributed on the cellsurface of various organs and are present in burns, wounds, dermis andtumors. The peptide targeting moiety may also be a cyclic peptidecontaining within its sequence a RGD integrin binding motif. Ligandssuch as the RGD peptide that bind to integrins on the cell surface canmediate internalization, thus increasing the efficiency of delivery ofthe targeted complex. The targeting peptide may include an RGDFV (SEQ IDNO:3) sequence, wherein the peptide includes the RGD sequence in whichthe peptide is from 3 to 30 amino acids in length. In other embodimentsthe RGD integrin binding motif is from 3 to 20 amino acids in length or4 to 10 amino acids in length. In particular embodiments of the presentinvention, the RGD integrin binding motif is a peptide 5 amino acids inlength. Although cyclic peptides which contain the RGD integrin bindingmotif within its sequence are preferred, linear peptides may also beutilized in the present invention. 20060239968 Compositions and methodsof use of targeting peptides for diagnosis and therapy of human cancer.U.S. Patent publications 20060094672, 20050191294, 20050187161,20050074812, 20050074747, 20050037417, 20050003466, 20040170955,20040131623, 20040096441, 20040071689, 20040048243, 20030152578,20030113320, and 20010046498, as well as PCT publications WO2006/060171, WO 2006/010070, WO 2005/065418, WO 2005/026195, WO2004/020999, WO 2003/022991, WO 2002/020822, WO 2002/020769, WO2002/020723, and WO 2002/020722, describe various compositions andmethods of identifying targeting ligands, each of which is incorporatedherein by reference in its entirety.

As used herein, “ligand” refers to any peptide, polypeptide, protein ornon-protein, such as a peptidomimetic, that is capable of binding to acell-surface molecule and internalizing. As used herein, to “bind to areceptor” refers to the ability of a ligand to specifically recognizeand detectably bind to a receptor, as assayed by standard in vitro or invivo assays.

Within the context of this invention, the ligand is coupled to a proteinof a phage (e.g., a capsid protein), either as a fusion protein orthrough chemical conjugation, and is used to deliver a nucleic acid to acell. Fragments of ligands may be used within the present invention, solong as the fragment retains the ability to bind to the appropriate cellsurface molecule. Likewise, ligands with substitutions or otheralterations, but which retain binding ability, may also be used. Aswell, a particular ligand refers to a polypeptide(s) having an aminoacid sequence of the native ligand, as well as modified sequences,(e.g., having amino acid substitutions, deletions, insertions oradditions compared to the native protein (muteins)) as long as theligand retains the ability to bind to its receptor on an endothelialcell and result in delivery of a nucleic acid to a cell.

Ligands also encompass muteins or mutant proteins that possess theability to bind to its receptor expressing cells and be internalized.Such muteins include, but are not limited to, those produced byreplacing one or more of the cysteines with serine. Typically, suchmuteins will have conservative amino acid changes. DNA encoding suchmuteins will, unless modified by replacement of degenerate codons,hybridize under conditions of at least low stringency to native DNAsequence encoding the wild-type ligand.

DNA encoding a ligand may be prepared synthetically based on known aminoacid or DNA sequence, isolated using methods known to those of skill inthe art (e.g., PCR amplification), or obtained from commercial or othersources. DNA encoding a ligand may differ from the above sequences bysubstitution of degenerate codons or by encoding different amino acids.Differences in amino acid sequences, such as those occurring among thehomologous ligand of different species as well as among individualorganisms or species, are tolerated as long as the ligand binds to itsreceptor. Ligands may be isolated from natural sources or madesynthetically, such as by recombinant means or chemical synthesis.

It is not necessary that the ligands used in the context of thisinvention retain any of its in vivo biological activities, other thanbinding a receptor on a cell and be internalized. If the ligand has beenmodified so as to lack one or more biological activities, binding andinternalization may still be readily assayed, for example, by thefollowing tests or other tests known in the art. Generally, these testsinvolve labeling the ligand, incubating it with target cells, andvisualizing or measuring intracellular label. For example, briefly, theligand may be fluorescently labeled with FITC or radiolabeled with ¹²⁵I,incubated with cells and examined microscopically by fluorescencemicroscopy or confocal microscopy for internalization.

The ligands may be produced by recombinant or other means in preparationfor attachment to phage capsid proteins. The DNA sequences and methodsto obtain the sequences of these ligands are well known. Based on theDNA sequences, the genes may be synthesized either synthetically (forsmall proteins), amplified from cell genomic or cDNA, isolated fromgenomic or cDNA libraries and the like. Restriction sites to facilitatecloning into the phage or phagemid vector may be incorporated, such asin primers for amplification.

Such molecules include, without limitation, proteins that bind cancercells, endothelial cells, stromal cells, and the like. Such ligandsinclude growth factors and cytokines Many growth factors and families ofgrowth factors share structural and functional features and may be usedin the present invention. Families of growth factors include fibroblastgrowth factors FGF-1 through FGF-15, and vascular endothelial growthfactor (VEGF). Other growth factors, such as PDGF (platelet-derivedgrowth factor), TGF-α (transforming growth factor), TGF-β, HB-EGF,angiotensin, bombesis, erythopoietin, stem cell factor, M-CSF, G-CSF,GM-CSF, and endoglin also bind to specific identified receptors on cellsurfaces and may be used in the present invention. Cytokines, includinginterleukins, CSFs (colony stimulating factors), and interferons, havespecific receptors, and may be used as described herein.

For example, ligands and ligand/receptor pairs includeurokinase/urokinase receptor (GenBank Accession Nos. X02760/X74309);α-1,3 fucosyl transferase, α1-antitrypsin/E-selectin (GenBank AccessionNos. M98825, D38257/M87862); P-selectin glycoprotein ligand, P-selectinligand/P-selectin (GenBank Accession Nos. U25955, U02297/L23088),VCAM1/VLA-4 (GenBank Accession Nos. X53051/X16983); E9 antigen (Blann etal., Atherosclerosis 120:221, 1996)/TGFβ receptor; Fibronectin (GenBankAccession No. X02761); type I α1-collagen (GenBank Accession No.Z74615), type I β2-collagen (GenBank Accession No. Z74616), hyaluronicacid/CD44 (GenBank Accession No. M59040); CD40 ligand (GenBank AccessionNo. L07414)/CD40 (GenBank Accession No. M83312); EFL-3, LERTK-2 ligands(GenBank Accession Nos. L37361, U09304) for elk-1 (GenBank Accession No.M25269); VE-cadherin (GenBank Accession No. X79981); ligand forcatenins; ICAM-3 (GenBank Accession No. X69819) ligand for LFA-1, andvon Willebrand Factor (GenBank Accession No. X04385), fibrinogen andfibronectin (GenBank Accession No. X92461) ligands for α_(v)β₃ integrin(GenBank Accession Nos. U07375, L28832).

Other ligands include CSF-1 (GenBank Accession Nos. M11038, M37435);GM-CSF (GenBank Accession No. X03021); IFN-α (interferon) (GenBankAccession No. A02076; WO 8502862-A); IFN-γ (GenBank Accession No.A02137; WO 8502624-A); IL-1-β (interleukin 1 alpha) (GenBank AccessionNo. X02531, M15329); IL-1-β (interleukin 1 beta) (GenBank Accession No.X02532, M15330, M15840); IL-1 (GenBank Accession No. K02770, M54933,M38756); IL-2 (GenBank Accession No. A14844, A21785, X00695, X00200,X00201, X00202); IL-3 (GenBank Accession No. M14743, M20137); IL-4(GenBank Accession No. M13982); IL-5 (GenBank Accession No. X04688,J03478); IL-6 (GenBank Accession No. Y00081, X04602, M54894, M38669,M14584); IL-7 (GenBank Accession No. J04156); IL-8 (GenBank AccessionNo. Z11686); IL-10 (GenBank Accession No. X78437, M57627); IL-11(GenBank Accession No. M57765 M37006); IL-13 (GenBank Accession No.X69079, U10307); TNF-α (Tumor necrosis factor) (GenBank Accession No.A21522); TNF-β (GenBank Accession No. D12614); GP30 ligand (S68256) forerbB2; and transferrin (GenBank Accession No. DQ923758) for thetransferrin receptor.

Still other ligands include PDGF (GenBank Accession No. X03795, X02811),angiotensin (GenBank Accession No. K02215), and all RGD-containingpeptides and proteins, such as ICAM-1 (GenBank Accession No. X06990) andVCAM-1 (GenBank Accession No. X53051) that bind to integrin receptors.Other ligands include TNFα (GenBank Accession No. A21522, X01394), IFN-γ(GenBank Accession No. A11033, A11034), IGF-I (GenBank Accession No.A29117, X56773, 561841, X56774, S61860), IGF-II (GenBank Accession No.A00738, X06159, Y00693), atrial naturietic peptide (GenBank AccessionNo. X54669), endothelin-1 (GenBank Accession No. Y00749), coagulationfactor Xa (GenBank Accession No. L00395, L00396, L29433, N00045,M14327), TGF-β1 (GenBank Accession No. A23751), IL-1β (GenBank AccessionNo. X03833), IL-1β (GenBank Accession No. M15330), and endoglin (GenBankAccession No. X72012).

The family of FGF proteins presently includes FGF-1 (acidic FGF oraFGF), FGF-2 (basic FGF or bFGF), FGF-3 (int-2), FGF-4 (hst-1/K-FGF),FGF-5, FGF-6 (hst-2), FGF-7 (keratinocyte growth factor or KGF), FGF-8,FGF-9, FGF-11 (WO 96/39507), FGF-13 (WO 96/39508), FGF-14 (WO 96/39506),and FGF-15 (WO 96/39509). Other polypeptides that are reactive with anFGF receptor, that is any polypeptide that specifically interacts withan FGF receptor, preferably the high affinity FGF receptor, and istransported by way of endosomes into the cell by virtue of itsinteraction with the FGF receptor are suitable within the presentinvention. Ligands also include 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 ormore amino acid fragments of the ligands identified herein.

Also contemplated as targeting moieties are antibodies and fragmentsthereof. Monoclonal antibody fragments may be used to target delivery tospecific organs in the animal including brain, heart, lungs or liver. Anexemplary method for targeting viral particles to cells that lack asingle cell-specific marker is described (U.S. Pat. No. 5,849,718). Forexample, antibody A may have specificity for tumor, but also for normalheart and lung tissue, while antibody B has specificity for tumor butalso normal liver cells. Clearly, the use of antibody A or antibody Balone to deliver an anti-proliferative nucleic acid to the tumor wouldpossibly result in unwanted damage to heart and lung or liver cells.However, antibody A and antibody B can be used together for improvedcell targeting. Thus, antibody A is coupled to a gene encoding ananti-proliferative nucleic acid and is delivered, via a receptormediated uptake system, to tumor as well as heart and lung tissue.However, the gene is not transcribed or active in these cells. AntibodyB can be coupled to an activator or a universally active gene encoding afactor necessary for the transcription or activation of theanti-proliferative nucleic acid and is delivered to tumor and livercells. Therefore, in heart and lung cells only the inactiveanti-proliferative nucleic acid is delivered, where it is nottranscribed, leading to no adverse effects. In liver cells, the geneencoding the activation factor is delivered, but has no effect becauseno an anti-proliferative nucleic acid gene is present. In tumor cells,however, both genes are delivered and the activation factor can activatethe anti-proliferative nucleic acid, leading to tumor-specific toxiceffects.

Antibodies to molecules expressed on the surface of cells are usefulwithin the context of the present invention. Such antibodies include,but are not limited to, antibodies to FGF receptors, VEGF receptors,urokinase receptor, E- and P-selectins, VCAM-1, PDGF receptor, TGFreceptor, endosialin, α_(v)β₃ integrin, LFA-1, E9 antigen, CD40,cadherins, and elk-1. Antibodies that are specific to cell surfacemolecules expressed by cells are readily generated as monoclonals orpolyclonal antisera. Many such antibodies are available (e.g., fromAmerican Type Culture Collection, Rockville, Md.). Alternatively,antibodies to ligands that bind/internalize may also be used. In such astrategy, the phage particles will have antibody on their surface, whichwill then be complexed to the ligand.

Many other ligands may be employed for the targeting step of AAVPpreparations, depending upon the site targeted for AAVP delivery. Incertain embodiments, it is contemplated that AAVP are targeted tospecific cell types by receptor-mediated endocytosis. For example,lactosyl ceramide, and peptides that target the LDL receptor relatedproteins, such as apolipoprotein E3 (“Apo E”) have been useful intargeting liposomes to the liver (Spanjer and Scherphof, 1983; WO98/0748). The asialoglycoprotein, asialofetuin, which contains terminalgalactosyl residues, also has been demonstrated to target liposomes tothe liver (Spanjer and Scherphof, 1983; Hara et al., 1995). The sugarsmannosyl, fucosyl or N-acetyl glucosamine, when coupled to the backboneof a polypeptide, bind the high affinity manose receptor (U.S. Pat. No.5,432,260, specifically incorporated herein by reference in itsentirety). Thus, these glycoproteins can be conjugated to AAVP of thepresent invention and are contemplated as useful for targeting specificcells (e.g., macrophages).

Folate and the folate receptor have also been described as useful forcellular targeting (U.S. Pat. No. 5,871,727). In this example, thevitamin folate is coupled to the AAVP capsid protein(s). The folatereceptor has high affinity for its ligand and is overexpressed on thesurface of several malignant cell lines, including lung, breast andbrain tumors. Transferrin mediated delivery systems target a wide rangeof replicating cells that express the transferrin receptor (Gilliland etal., 1980).

The addition of targeting ligands for gene delivery for the treatment ofhyperproliferative diseases permits the delivery of genes whose geneproducts are more toxic than do non-targeted systems. Examples of themore toxic genes that can be delivered includes pro-apoptotic genes suchas Bax and Bak plus genes derived from viruses and other pathogens suchas the adenoviral E4orf4 and the E. coli purine nucleosidephosphorylase, a so-called “suicide gene” which converts the prodrug6-methylpurine deoxyriboside to toxic purine 6-methylpurine. Otherexamples of suicide genes used with prodrug therapy are the E. colicytosine deaminase gene and the HSV thymidine kinase gene.

In certain aspects, AAVP can be used to target tumor vasculature. Tumorendothelium is an important target for cancer therapy. Targeting atherapeutic gene of interest to the tumor endothelium with minimaltoxicity in other tissues remains the primary goal of antivascular genetherapy. Recently, AAVP targeting tumor endothelium have been described.The inventors studied the ability of this vector to deliver a potentantivascular agent, human tumor necrosis factor-α (TNF-α) to humanmelanomas. TNF-α resistant melanoma was made sensitive to TNF-αtreatment by delivering endothelial monocyte activating polypeptide-II(EMAP-II) via AAVP.

AAVP vectors carrying two genes, TNF-α and EMAP-II were evaluated invitro and in vivo. Human melanoma cells (M21) were studied for AAVPinternalization and TNF-α gene expression in vitro. M21/Pmelsubcutaneously grown tumors in nude mice were treated systemicallythrough tail vein injections. The localization of targeted AAVP to thetumor vasculature, TNF-α gene expression and apoptosis were examinedusing immunofluorescence staining, TaqMan RT-PCR and immunohistochemicalanalysis.

Internalization of targeted AAVP was observed in M21 cells, resulting inhigh levels of functionally active TNF-α in the culture supernatant. Nointernalization of non-targeted vector was observed in these cells.Systemic injection of AAVP showed tumor targeted virus delivery withminimal virus localization into normal organs. The AAVP deliveryresulted in expression of TNF-α gene product. The expression of TNF-α,induced apoptosis in the blood vessels and surrounding tumor cellsresulting in significant tumor regression. Additionally, targeteddelivery of AAVP expressing EMAP-II sensitized a TNF-α resistant humanmelanoma to TNF-α treatment. Targeted AAVP vectors can be used todeliver antivascular agents specifically to tumor vasculature, thusreducing the systemic toxicity.

The AAVP of the invention can be targeted to specific regions of thebody by attachment of specific targeting ligands to provide rapidaccumulation and concentration of AAVP and, correspondingly, of nucleicacid molecules, in a designated tissue. The ligands contemplated for usein the present invention can be conjugated to the AAVP by a variety ofmethods. Various compositions and methods for coupling a targetingligand to a capsid protein are known in the art.

II. NUCLEIC ACIDS

The AAVP of the present disclosure have the ability to deliver one ormore transgenes to the nucleus of the target cell, thereby enhancing theexpression of the transgene in the target cell. An AAVP may also bestowan advantage in gene expression by means of an altered fate of thetransgene cassette through formation of concatamers of the transgenecassette, thereby leading to enhanced gene expression.

The term “transgene,” as used herein, refers to a gene or geneticmaterial that has been transferred from one organism to another. Atransgene may comprise one or more genes and/or one or moreoligonucleotides. For example, a transgene may comprise a reporter gene,a suicide gene, a prodrug converting enzyme, and/or one or moretherapeutic genes. As used herein, “oligonucleotide” refers to a shortnucleic acid sequence with twenty or fewer base pairs. The term“therapeutic gene,” as used herein, is defined as a nucleic acid region,which provides a therapeutic effect on a disease, medical condition,organ, tissue, cell or physiologic characteristic of an organism. Asused herein, the term “cassette” as used herein is a nucleic acid whichcan express a protein, polypeptide, or RNA of interest.

In addition to ligands an AAVP may contain a transgene comprising areporter gene whose product can be selected for or detected. As referredto herein, a “reporter gene” is a nucleic acid region that encodes for aproduct that can be detected, such as by fluorescence, enzyme activityon a detectably labeled compound or chromogenic substrate, orfluorescent substrate, and the like; or selected for by growthconditions. Such reporter genes include, without limitation, greenfluorescent protein (GFP), β-galactosidase, chloramphenicolacetyltransferase (CAT), luciferase, neomycin phosphotransferase,secreted alkaline phosphatase (SEAP), human growth hormone (HGH),thymidine kinase, and the like. Selectable markers include drugresistances, such as neomycin (G418), hygromycin, and the like. Incertain embodiments, a reporter gene that encodes for a secreted proteinthat can be detected in blood, other bodily fluids, or tissues may beused to measure the level expression of the reporter gene.

In addition to a reporter gene, the transgene also may comprise atherapeutic gene. AAVPs carrying such transgenes may allow for, amongother things, imaging a subject (e.g., a human), either in vitro or invivo. In vitro imaging may allow for non-invasive imaging of the wholesubject or of target areas of the subject. After introduction of thesetransgenes, expression may be imaged using imaging techniques known inthe art (e.g., BLI imaging, PET imaging, fluorescent imaging, and thelike.) A “subject,” as used herein, refers to any mammalian entity, forexample, a subject may be an human in need of gene therapy or othertreatment.

In other embodiments, the AAVP may contain a suicide gene. The term“suicide gene” as used herein is defined as a nucleic acid which, uponadministration of a prodrug, effects transition of a gene product to acompound which kills its host cell. Examples of suicide gene/prodrugcombinations which may be used are Herpes Simplex Virus-thymidine kinase(HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase andcycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinasethymidilate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase andcytosine arabinoside. In certain embodiments, a suicide gene may act inthe manner of a therapeutic gene by providing a therapeutic effect on adisease or medical condition as a result of the killing of its hostcell.

The term “nucleic acid” is well known in the art. A “nucleic acid” asused herein will generally refer to a molecule (i.e., a strand) of DNA,RNA or a derivative or analog thereof, comprising a nucleobase. Anucleobase includes, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g., an adenine “A,” a guanine “G,” athymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil “U” ora C). The term “nucleic acid” encompass the terms “oligonucleotide” and“polynucleotide,” each as a subgenus of the term “nucleic acid.” Theterm “oligonucleotide” refers to a molecule of between about 8 and about100 nucleobases in length. The term “polynucleotide” refers to at leastone molecule of greater than about 100 nucleobases in length.

Herein certain embodiments, a “gene” refers to a nucleic acid that istranscribed. In certain aspects, the gene includes regulatory sequencesinvolved in transcription, or message production or composition. As willbe understood by those in the art, this functional term “gene” includesboth genomic sequences, RNA or cDNA sequences or smaller engineerednucleic acid segments, including nucleic acid segments of anon-transcribed part of a gene, including but not limited to thenon-transcribed promoter or enhancer regions of a gene. Smallerengineered gene nucleic acid segments may express, or may be adapted toexpress using nucleic acid manipulation technology, proteins,polypeptides, domains, peptides, fusion proteins, mutants and/or suchlike.

A polynucleotide of the invention may form an “expression cassette.” An“expression cassette” is polynucleotide that provides for the expressionof a particular transcription unit. That is it includes promoterelements and various other elements that function in the transcriptionof a gene or transcription unit. An expression cassette may also be partof a larger replicating polynucleotide or expression vector orconstruct.

These definitions generally refer to a single-stranded molecule, but inspecific embodiments will also encompass an additional strand that ispartially, substantially or fully complementary to the single-strandedmolecule. Thus, a nucleic acid may encompass a double-stranded moleculethat comprises one or more complementary strand(s) or “complement(s)” ofa particular sequence comprising a molecule.

“Isolated substantially away from other coding sequences” means that thegene of interest forms the significant part of the coding region of thenucleic acid, or that the nucleic acid does not contain large portionsof naturally-occurring coding nucleic acids, such as large chromosomalfragments, other functional genes, RNA or cDNA coding regions. Ofcourse, this refers to the nucleic acid as originally isolated, and doesnot exclude genes or coding regions later added to the nucleic acid bythe hand of man.

As used herein a “nucleobase” refers to a heterocyclic base, such as forexample a naturally occurring nucleobase (i.e., an A, T, G, C or U)found in at least one naturally occurring nucleic acid (i.e., DNA andRNA), and naturally or non-naturally occurring derivative(s) and analogsof such a nucleobase. A nucleobase generally can form one or morehydrogen bonds (“anneal” or “hybridize”) with at least one naturallyoccurring nucleobase in manner that may substitute for naturallyoccurring nucleobase pairing (e.g., the hydrogen bonding between A andT, G and C, and A and U).

As used herein, a “nucleotide” refers to a nucleoside further comprisinga “backbone moiety.” A backbone moiety generally covalently attaches anucleotide to another molecule comprising a nucleotide, or to anothernucleotide to form a nucleic acid. The “backbone moiety” in naturallyoccurring nucleotides typically comprises a phosphorus moiety, which iscovalently attached to a 5-carbon sugar. The attachment of the backbonemoiety typically occurs at either the 3′- or 5′-position of the 5-carbonsugar. However, other types of attachments are known in the art,particularly when a nucleotide comprises derivatives or analogs of anaturally occurring 5-carbon sugar or phosphorus moiety.

A. Expression Constructs

Expression constructs of the invention may include nucleic acidsencoding a therapeutic nucleic acid and/or imaging protein. In otheraspects, the expression construct may be a therapeutic expressionconstruct that can be used in therapeutic compositions and methods ofthe invention. In certain embodiments, genetic material may bemanipulated to produce expression cassettes and/or expression constructsthat encode imaging proteins, targeting proteins and/or therapeuticgenes.

Embodiments of the invention may include two separate types ofexpression cassette or expression construct comprising an expressioncassette. One cassette is used in expression of an imaging protein,i.e., a protein that is directly detectable or has an activity ofproperty that is indirectly detectable. Another expression cassette mayencode a therapeutic gene. In the context of a therapeutic vector, atherapeutic gene may be a therapeutic gene discussed herein useful inthe prophylatic or therapeutic treatment of a disease condition. In thecontext of gene therapy, the gene may be a heterologous DNA, meant toinclude DNA derived from a source other than the viral genome whichprovides the backbone of the vector. The gene may be derived from aprokaryotic or eukaryotic source such as a bacterium, a virus, a yeast,a parasite, a plant, or even an animal. The heterologous DNA also may bederived from more than one source, i.e., a multigene construct or afusion protein. The heterologous DNA also may include a regulatorysequence which may be derived from one source and the gene from adifferent source.

B. Control Regions

Expression cassettes and/or constructs of the invention, whether theyencode an imaging protein or a therapeutic gene(s) will typicallyinclude various control regions. These control region typically modulatethe expression of the gene of interest.

1. Promoters

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid codingfor gene products in which part or all of the nucleic acid encodingsequence is capable of being transcribed, e.g., all or part of animaging protein or therapeutic protein. The transcript may be translatedinto a protein, but it need not be. In certain embodiments, expressionincludes both transcription of a gene and translation of mRNA into agene product. In other embodiments, expression only includestranscription of a therapeutic nucleic acid such as inhibitory RNAs orDNAs.

The nucleic acid encoding a gene product is under transcriptionalcontrol of a promoter. A “promoter” refers to a DNA sequence recognizedby the machinery of the cell, or introduced machinery, required toinitiate the specific transcription of a gene. In particular aspects,transcription may be constitutive, inducible, and/or repressible. Thephrase “under transcriptional control” means that the promoter is in thecorrect location and orientation in relation to the nucleic acid tocontrol RNA polymerase initiation and expression of the gene.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for various retroviral promoters, the HSV thymidine kinase (tk)and SV40 early transcription units.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. Depending on the promoter, it appearsthat individual elements can function either co-operatively orindependently to activate transcription.

The particular promoter employed to control the expression of a nucleicacid sequence of interest is not believed to be important, so long as itis capable of directing the expression of the nucleic acid in thetargeted cell. Thus, where a human cell is targeted, it is preferable toposition the nucleic acid coding region adjacent to and under thecontrol of a promoter that is capable of being expressed in a targetedhuman cell. Generally speaking, such a promoter might include either ahuman, viral promoter or a combination thereof.

In various embodiments, the human cytomegalovirus immediate early genepromoter (CMVIE), the SV40 early promoter, the Rous sarcoma virus longterminal repeat, β-actin, rat insulin promoter andglyceraldehyde-3-phosphate dehydrogenase can be used to obtainhigh-level expression of the coding sequence of interest. The use ofother viral, retroviral or mammalian cellular or bacterial phagepromoters, which are well-known in the art to achieve expression of acoding sequence of interest is contemplated as well, provided that thelevels of expression are sufficient for a given purpose. By employing apromoter with well-known properties, the level and pattern of expressionof the protein of interest following transfection or transformation canbe optimized.

Selection of a promoter that is regulated in response to specificphysiologic or synthetic signals can permit inducible expression of thegene product as compared with the cell under non-inducing conditions.For example in the case where expression of a transgene, or transgeneswhen a multicistronic vector is utilized, is toxic to the cells in whichthe vector is produced in, it may be desirable to prohibit or reduceexpression of one or more of the transgenes. Examples of transgenes thatmay be toxic to the producer cell line are pro-apoptotic and cytokinegenes. Several inducible promoter systems are available for productionof viral vectors where the transgene product may be toxic.

The ecdysone system (Invitrogen, Carlsbad, Calif.) is one such system.This system is designed to allow regulated expression of a gene ofinterest in mammalian cells. It consists of a tightly regulatedexpression mechanism that allows virtually no basal level expression ofthe transgene, but over 200-fold inducibility. Another inducible systemthat would be useful is the Tet-Off™ or Tet-On™ system (Clontech, PaloAlto, Calif.) originally developed by Gossen and Bujard (Gossen andBujard, 1992; Gossen et al., 1995). This system also allows high levelsof gene expression to be regulated in response to tetracycline ortetracycline derivatives such as doxycycline.

In some circumstances, it may be desirable to regulate expression of atransgene in a therapeutic expression vector. For example, differentviral promoters with varying strengths of activity may be utilizeddepending on the level of expression desired. In mammalian cells, theCMV immediate early promoter if often used to provide strongtranscriptional activation. Modified versions of the CMV promoter thatare less potent have also been used when reduced levels of expression ofthe transgene are desired. When expression of a transgene inhematopoietic cells is desired, retroviral promoters such as the LTRsfrom MLV or MMTV are often used. Other viral promoters that may be useddepending on the desired effect include SV40, RSV LTR, HIV-1 and HIV-2LTR, adenovirus promoters such as from the E1A, E2A, or MLP region, AAVLTR, cauliflower mosaic virus, HSV-TK, and avian sarcoma virus.

Similarly tissue specific or selective promoters may be used to effecttranscription in specific tissues or cells so as to reduce potentialtoxicity or undesirable effects to non-targeted tissues. For example,promoters such as the PSA, probasin, prostatic acid phosphatase orprostate-specific glandular kallikrein (hK2) may be used to target geneexpression in the prostate. Similarly, the following promoters may beused to target gene expression in other tissues (Table 1).

In certain indications, it may be desirable to activate transcription atspecific times after administration of the gene therapy vector. This maybe done with such promoters as those that are hormone or cytokineregulatable. For example in therapeutic applications where theindication is a gonadal tissue where specific steroids are produced orrouted to, use of androgen or estrogen regulated promoters may beadvantageous. Such promoters that are hormone regulatable include MMTV,MT-1, ecdysone and RuBisco. Other hormone regulated promoters such asthose responsive to thyroid, pituitary and adrenal hormones are expectedto be useful in the present invention. Cytokine and inflammatory proteinresponsive promoters that could be used include K and T Kininogen(Kageyama et al., 1987), c-fos, TNF-alpha, C-reactive protein (Arcone etal., 1988), haptoglobin (Oliviero et al., 1987), serum amyloid A2, C/EBPalpha, IL-1, IL-6 (Poli and Cortese, 1989), Complement C3 (Wilson etal., 1990), IL-8, alpha-1 acid glycoprotein (Prowse and Baumann, 1988),alpha-1 antitypsin, lipoprotein lipase (Zechner et al., 1988),angiotensinogen (Ron et al., 1990), fibrinogen, c-jun (inducible byphorbol esters, TNF-alpha, UV radiation, retinoic acid, and hydrogenperoxide), collagenase (induced by phorbol esters and retinoic acid),metallothionein (heavy metal and glucocorticoid inducible), Stromelysin(inducible by phorbol ester, interleukin-1 and EGF), alpha-2macroglobulin and alpha-1 antichymotrypsin. Tumor specific promoterssuch as osteocalcin, hypoxia-responsive element (HRE), MAGE-4, CEA,alpha-fetoprotein, GRP78/BiP and tyrosinase may also be used to regulategene expression in tumor cells.

It is envisioned that any of the above promoters alone or in combinationwith another may be useful according to the present invention dependingon the action desired. In addition, this list of promoters should not beconstrued to be exhaustive or limiting, those of skill in the art willknow of other promoters that may be used in conjunction with thepromoters and methods disclosed herein.

TABLE 1 TISSUE SPECIFIC PROMOTERS Tissue Promoter Pancreas InsulinElastin Amylase pdr-1 pdx-1 glucokinase Liver Albumin PEPCK HBV enhancerα fetoprotein apolipoprotein C α-1 antitrypsin vitellogenin, NF-ABTransthyretin Skeletal muscle Myosin H chain Muscle creatine kinaseDystrophin Calpain p94 Skeletal alpha-actin fast troponin 1 Skin KeratinK6 Keratin K1 Lung CFTR Human cytokeratin 18 (K18) Pulmonary surfactantproteins A, B and C CC-10 P1 Smooth muscle sm22 α SM-alpha-actinEndothelium Endothelin-1 E-selectin von Willebrand factor TIE (Korhonenet al., 1995) KDR/flk-1 Melanocytes Tyrosinase Adipose tissueLipoprotein lipase (Zechner et al., 1988) Adipsin (Spiegelman et al.,1989) acetyl-CoA carboxylase (Pape and Kim, 1989) glycerophosphatedehydrogenase (Dani et al., 1989) adipocyte P2 (Hunt et al., 1986) Bloodβ-globin

2. Enhancers

Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Enhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins. The basic distinction between enhancers andpromoters is operational. An enhancer region as a whole must be able tostimulate transcription at a distance; this need not be true of apromoter region or its component elements. On the other hand, a promotermust have one or more elements that direct initiation of RNA synthesisat a particular site and in a particular orientation, whereas enhancerslack these specificities. Promoters and enhancers are often overlappingand contiguous, often seeming to have a very similar modularorganization.

Below is a list of promoters additional to the tissue specific promoterslisted above, cellular promoters/enhancers and induciblepromoters/enhancers that could be used in combination with the nucleicacid encoding a gene of interest in an expression construct (Table 2 andTable 3). Additionally, any promoter/enhancer combination (as per theEukaryotic Promoter Data Base EPDB) could also be used to driveexpression of the gene. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct.

In preferred embodiments of the invention, a therapeutic expressionconstruct comprises a virus or engineered construct derived from a viralgenome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis and to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden,1986; Temin, 1986).

TABLE 2 ENHANCER Immunoglobulin Heavy Chain Immunoglobulin Light ChainT-Cell Receptor HLA DQ α and DQ β β-Interferon Interleukin-2Interleukin-2 Receptor MHC Class II 5 MHC Class II HLA-DRα β-ActinMuscle Creatine Kinase Prealbumin (Transthyretin) Elastase IMetallothionein Collagenase Albumin Gene α-Fetoprotein τ-Globin β-Globine-fos c-HA-ras Insulin Neural Cell Adhesion Molecule (NCAM)α1-Antitrypsin H2B (TH2B) Histone Mouse or Type I CollagenGlucose-Regulated Proteins (GRP94 and GRP78) Rat Growth Hormone HumanSerum Amyloid A (SAA) Troponin I (TN I) Platelet-Derived Growth FactorDuchenne Muscular Dystrophy SV40 Polyoma Retroviruses Papilloma VirusHepatitis B Virus Human Immunodeficiency Virus Cytomegalovirus GibbonApe Leukemia Virus

TABLE 3 Element Inducer MT II Phorbol Ester (TPA) Heavy metals MMTV(mouse mammary tumor Glucocorticoids virus) β-Interferon Poly(rI)XPoly(rc) Adenovirus 5 E2 Ela c-jun Phorbol Ester (TPA), H₂O₂ CollagenasePhorbol Ester (TPA) Stromelysin Phorbol Ester (TPA), IL-1 SV40 PhorbolEster (TPA) Murine MX Gene Interferon, Newcastle Disease Virus GRP78Gene A23187 α-2-Macroglobulin IL-6 Vimentin Serum MHC Class I Gene H-2kBInterferon HSP70 Ela, SV40 Large T Antigen Proliferin Phorbol Ester-TPATumor Necrosis Factor FMA Thyroid Stimulating Hormone α Thyroid HormoneGene Insulin E Box Glucose

C. Polyadenylation Signals

Polyadenylation signals may be used in therapeutic and/or imagingvectors. Where a cDNA insert is employed, one will typically desire toinclude a polyadenylation signal to effect proper polyadenylation of thegene transcript. The nature of the polyadenylation signal is notbelieved to be crucial to the successful practice of the invention, andany such sequence may be employed such as human or bovine growth hormoneand SV40 polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

D. Therapeutic Genes

AAVP particles of the invention may be used to deliver a variety oftherapeutic or imaging agents, including therapeutic expression vectors.The present invention contemplates the use of a variety of differenttherapeutic genes. For example, genes encoding enzymes, hormones,cytokines, oncogenes, receptors, ion channels, tumor suppressors,transcription factors, drug selectable markers, toxins and variousantigens are contemplated as suitable genes for use according to thepresent invention. In addition, antisense and inhibitory RNA constructsderived from oncogenes are other “genes” of interest according to thepresent invention.

In accordance with the present invention, a selected gene or polypeptidemay refer to any protein, polypeptide, or peptide. A therapeutic gene orpolypeptide is a gene or polypeptide which can be administered to asubject for the purpose of treating or preventing a disease. Forexample, a therapeutic gene can be a gene administered to a subject fortreatment or prevention of cancer. Examples of therapeutic genesinclude, but are not limited to, Rb, CFTR, p16, p21, p27, p57, p73,C-CAM, APC, CTS-1, zac1, scFV ras, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II,BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF, G-CSF, thymidine kinase,Bax, Bak, Bik, Bim, Bid, Bad, Harakiri, Fas-L, mda-7, fus, interferon α,interferon β, interferon γ, ADP, p53, ABLI, BLC1, BLC6, CBFA1, CBL,CSFIR, ERBA, ERBB, EBRB2, ETS1, ETS2, ETV6, FGR, FOX, FYN, HCR, HRAS,JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML,RET, SRC, TAL1, TCL3, YES, MADH4, RB1, TP53, WT1, TNF, BDNF, CNTF, NGF,IGF, GMF, aFGF, bFGF, NT3, NT5, ApoAI, ApoAIV, ApoE, Rap1A, cytosinedeaminase, Fab, ScFv, BRCA2, zac1, ATM, HIC-1, DPC-4, FHIT, PTEN, ING1,NOEY1, NOEY2, OVCA1, MADR2, 53BP2, IRF-1, zac1, DBCCR-1, rks-3, COX-1,TFPI, PGS, Dp, E2F, ras, myc, neu, raf, erb, fms, trk, ret, gsp, hst,abl, E1A, p300, VEGF, FGF, thrombospondin, BAI-1, GDAIF, or MCC.

Other examples of therapeutic genes include genes encoding enzymes.Examples include, but are not limited to, ACP desaturase, an ACPhydroxylase, an ADP-glucose pyrophorylase, an ATPase, an alcoholdehydrogenase, an amylase, an amyloglucosidase, a catalase, a cellulase,a cyclooxygenase, a decarboxylase, a dextrinase, an esterase, a DNApolymerase, an RNA polymerase, a hyaluron synthase, a galactosidase, aglucanase, a glucose oxidase, a GTPase, a helicase, a hemicellulase, ahyaluronidase, an integrase, an invertase, an isomerase, a kinase, alactase, a lipase, a lipoxygenase, a lyase, a lysozyme, apectinesterase, a peroxidase, a phosphatase, a phospholipase, aphosphorylase, a polygalacturonase, a proteinase, a peptidease, apullanase, a recombinase, a reverse transcriptase, a topoisomerase, axylanase, a reporter gene, an interleukin, or a cytokine.

Further examples of therapeutic genes include the gene encodingcarbamoyl synthetase I, ornithine transcarbamylase, arginosuccinatesynthetase, arginosuccinate lyase, arginase, fumarylacetoacetatehydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin,glucose-6-phosphatase, low-density-lipoprotein receptor, porphobilinogendeaminase, factor VIII, factor IX, cystathione beta.-synthase, branchedchain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase,propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoAdehydrogenase, insulin, -glucosidase, pyruvate carboxylase, hepaticphosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein,T-protein, Menkes disease copper-transporting ATPase, Wilson's diseasecopper-transporting ATPase, cytosine deaminase, hypoxanthine-guaninephosphoribosyltransferase, galactose-1-phosphate uridyltransferase,phenylalanine hydroxylase, glucocerbrosidase, sphingomyelinase,-L-iduronidase, glucose-6-phosphate dehydrogenase, HSV thymidine kinase,or human thymidine kinase.

Therapeutic genes also include genes encoding hormones. Examplesinclude, but are not limited to, genes encoding growth hormone,prolactin, placental lactogen, luteinizing hormone, follicle-stimulatinghormone, chorionic gonadotropin, thyroid-stimulating hormone, leptin,adrenocorticotropin, angiotensin I, angiotensin II, β-endorphin,β-melanocyte stimulating hormone, cholecystokinin, endothelin I,galanin, gastric inhibitory peptide, glucagon, insulin, lipotropins,neurophysins, somatostatin, calcitonin, calcitonin gene related peptide,β-calcitonin gene related peptide, hypercalcemia of malignancy factor,parathyroid hormone-related protein, parathyroid hormone-relatedprotein, glucagon-like peptide, pancreastatin, pancreatic peptide,peptide YY, PHM, secretin, vasoactive intestinal peptide, oxytocin,vasopressin, vasotocin, enkephalinamide, metorphinamide, alphamelanocyte stimulating hormone, atrial natriuretic factor, amylin,amyloid P component, corticotropin releasing hormone, growth hormonereleasing factor, luteinizing hormone-releasing hormone, neuropeptide Y,substance K, substance P, or thyrotropin releasing hormone.

In yet another embodiment, the heterologous gene may include asingle-chain antibody. Methods for the production of single-chainantibodies are well known to those of skill in the art. The skilledartisan is referred to U.S. Pat. No. 5,359,046, (incorporated herein byreference) for such methods. A single chain antibody is created byfusing together the variable domains of the heavy and light chains usinga short peptide linker, thereby reconstituting an antigen binding siteon a single molecule.

E. Multigene Constructs and IRES

In certain embodiments of the invention, the use of internal ribosomebinding sites (IRES) elements are used to create multigene polycistronicmessages (Pelletier and Sonenberg, 1988). IRES elements from two membersof the picanovirus family (polio and encephalomyocarditis) have beendescribed (Pelletier and Sonenberg, 1988), as well an IRES from amammalian message (Macejak and Sarnow, 1991). IRES elements can belinked to heterologous open reading frames. Multiple open reading framescan be transcribed together, each separated by an IRES, creatingpolycistronic messages. By virtue of the IRES element, each open readingframe is accessible to ribosomes for efficient translation. Multiplegenes can be efficiently expressed using a single promoter/enhancer totranscribe a single message. Any heterologous open reading frame can belinked to IRES elements. This includes genes for secreted proteins,multi-subunit proteins, encoded by independent genes, intracellular ormembrane-bound proteins and selectable markers. In this way, expressionof several proteins can be simultaneously engineered into a cell with asingle construct and a single selectable marker.

F. Preparation of Nucleic Acids

A nucleic acid may be made by any technique known to one of ordinaryskill in the art, such as for example, chemical synthesis, enzymaticproduction or biological production. A non-limiting example of anenzymatically produced nucleic acid include one produced by enzymes inamplification reactions such as PCR™ (see for example, U.S. Pat. No.4,683,202 and U.S. Pat. No. 4,682,195, each incorporated herein byreference), or the synthesis of an oligonucleotide described in U.S.Pat. No. 5,645,897, incorporated herein by reference. A non-limitingexample of a biologically produced nucleic acid includes a recombinantnucleic acid produced (i.e., replicated) in a living cell, such as arecombinant DNA vector replicated in bacteria (see for example, Sambrooket al. 2001, incorporated herein by reference).

III. PHARMACEUTICAL COMPOSITIONS AND ROUTES OF ADMINISTRATION

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions of the AAVP compositions(therapeutic compositions) in a form appropriate for the intendedapplication. Generally, this will entail preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender the compositions suitable for introduction into a patient.Aqueous compositions of the present invention comprise an effectiveamount of the AAVP or other agent dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. The phrase“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce adverse, allergic, orother untoward reactions when administered to an animal or a human.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknow in the art. Except insofar as any conventional media or agent isincompatible with the AAVP compositions of the present invention, itsuse as an imaging reagent or in therapeutic compositions iscontemplated. Supplementary active ingredients, such as otheranti-cancer agents, can also be incorporated into the compositions.Under ordinary conditions of storage and use, these preparations containa preservative to prevent growth of microorganisms. Intravenous vehiclesinclude fluid and nutrient replenishers. Preservatives includeantimicrobial agents, anti-oxidants, chelating agents and inert gases.The pH and exact concentration of the various components in thepharmaceutical are adjusted according to well-known parameters.

An effective amount of the composition is determined based on theintended goal, such as imaging and/or ameliorating a condition ordisease, such as cancer. The term “unit dose” refers to a physicallydiscrete unit suitable for use in a subject, each unit containing apredetermined quantity of the therapeutic composition calculated toproduce the desired response in association with its administration,i.e., the appropriate route and treatment regimen. The quantity to beadministered, both according to number of treatments and unit dose,depends on the subject to be treated, the state of the subject, and theprotection desired. Precise amounts of the therapeutic composition alsodepend on the judgment of the practitioner and are peculiar to eachindividual.

Also contemplated are combination compositions that contain two activeingredients. In particular, the present invention provides forcompositions that contain AAVP compositions and at least a secondtherapeutic, for example, an anti-neoplastic drug.

A. Parenteral Administration

The active compositions of the present invention may be formulated forparenteral administration, e.g., formulated for injection via theintravenous, intramuscular, sub-cutaneous, or even intraperitonealroutes. The preparation of an aqueous composition that contains a secondagent(s) as active ingredients will be known to those of skill in theart in light of the present disclosure. Typically, such compositions canbe prepared as injectables, either as liquid solutions or suspensions;solid forms suitable for using to prepare solutions or suspensions uponthe addition of a liquid prior to injection can also be prepared; andthe preparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial ad antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, theparticular methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.

To facilitate a better understanding of the present invention, thefollowing examples of specific embodiments are given. In no way shouldthe following examples be read to limit or define the entire scope ofthe invention.

IV. EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. The present examples, along with themethods described herein are presently representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the invention. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the scopeof the claims will occur to those skilled in the art.

Example 1 AAVP Targeting

A. Experimental Procedures

Design, Construction, and Generation of Targeted AAVP Particles. RGD-4Cphage and RGD-4C AAVP were engineered in a two-step process: generationof an intermediate (RGD-4C fUSE5-MCS) and subsequent production ofRGD-4C phage construct and RGD-4C AAVP. RGD-4C fUSES-MCS contained theoligonucleotide insert encoding the specific targeting peptide RGD-4Cand a fragment of the fMCS plasmid that had a multicloning site (MCS)for insertion of the eukaryotic expression cassette. RGD-4Cphage-derived fUSE5 DNA and phage-derived fMCS DNA were purified fromlysates of host E. coli (MC1061). We obtained the intermediate RGD-4CfUSE5-MCS by ligating a 5.4 kb BamHIISacII fragment of the RGD-4C FUSESplasmid to the 4.1 kb BamHI/SacII fragment of the fMCS plasmid. Next, wecreated a targeted AAVP-GFP by cloning the Pad fragment (2.8 kb) ofpAAV-eGFP plasmid (enhanced GFP; Stratagene) from ITR to ITR into thePstI site of RGD 4C fUSES-MSC. Briefly, pAAV was digested with Pad torelease a 2.8 kb fragment, which was blunted with DNA polymerase andcloned into the blunted PstI site of RGD-4C fUSES-MSC. To generate thetargeted phage constructs without ITRs, a 2.3 kb fragment locatedbetween the ITRs of pAAV-eGFP and containing pCMV-GFP and SV40 poly Awas released by EcoRI digestion, blunted with DNA polymerase, thencloned into the MCS of RGD-4C fUSES-MSC. To select cells expressing GFP,the BamHI-Sac1 fragment of the pQBI phosphoglycerate kinase-1 (PGK;QBIOgene) promoter and containing a GFPneo fusion sequence was cloned inthe NotI site of AAVP or control phage constructs to ensure that cellsexpressing GFP were G418-resistant. The GFPneo fragment of pQBI PGK wasreleased by BamHI and Sac1 digestion, and blunted with DNA polymerase;then phosphorylated linkers to NotI were added. After NotI digestion,the 1.57 kb GFPneo fragment was cloned into the NotI site of AAVP ornon-chimeric phage construct. Finally, to generate a targeted AAVPparticle carrying the gene for HSVtk or Luc the BamHI-NotI fragmentcontaining HSVtk or Luc was subcloned into BamHI-NotI site of pAAVplasmid to replace GFP. The ITR-HSVtk-ITR or ITR-Luc-ITR fragments wereremoved from pAAV-HSVtk and pAAV-Luc then inserted into RGD-4CfUSE5-MCS. Constructs were verified by DNA sequencing and restrictionanalysis, purified from the culture supernatant of host E. coli(MC1061), re-suspended in PBS and recentrifuged. Resulting supernatantswere titrated in E. Coli (k91Kan). Serial dilutions were plated onLuria-Bertani (LB) agar plates containing tetracycline and kanamycin andtransducing units (TU) were determined by colony counting.

Mammalian Cell Surface Binding and Internalization Assays. The inventorsused the biopanning and rapid analysis of selective interactive ligands(termed BRASIL) method (Giordano et al., 2001) to evaluate phage bindingto intact cells. In brief, KS1767 cells were detached withethylenediaminetetraacetate (EDTA) and resuspended in Dulbecco'smodified Eagle's medium (DMEM) containing 1% BSA at 4×10⁶ cells per ml.The cell suspension (50 μl) was incubated with 10⁹ TU of either RGD-4CAAVP or AAVP clones displaying scrambled versions of RGD-4C (CDCFGDCRC(SEQ ID NO:2), CDCGFDCRC (SEQ ID NO:3), CRCDGFCDC (SEQ ID NO:4)), mutantRGE-4C peptide, or non targeted control. After 2 h, the AAVP/cellmixture (aqueous phase) was transferred to the top of a non-miscibleorganic phase (200 μl solution in a 400 μl Eppendorf tube) consisting ofdibutyl phthalate:cyclohexane (9:1 [v:v], D=1.03 g/ml) and centrifugedat 10,000 g for 10 min at 4° C. The tube was then snap frozen in liquidnitrogen, the bottom of the tube was sliced off, and the cell-AAVPpellet was isolated and membrane-bound AAVP recovered (Giordano et al.,2001).

For cell internalization, KS1767 cells were grown in tissue chamberslides (Lab-Tek II Chamber Slide System; Nalge Nunc InternationalCorp.), washed twice with PBS, incubated with 10⁹ TU of RGD-4C AAVP orcontrol AAVP displaying scrambled versions of RGD-4C or RGE-4C in DMEMcontaining 1% BSA at 37° C., and washed with PBS to remove unbound AAVPafter 4 h incubation. Bound clones to cell membranes were chemicallyeluted by rinsing cells with 20 mM glycine (pH 2.3). Next, cells werewashed three times with PBS, fixed with PBS containing 4%paraformaldehyde (PFA) at RT for 15 min, washed with PBS, permeabilizedwith 0.2% Triton X-100, washed with PBS, and blocked with PBS containing1% BSA. Cells were then incubated with a 1:200 dilution of the primaryanti-M13 bacteriophage antibody (Amersham) in PBS containing 1% BSA atRT for 2 h, washed with PBS, and incubated with a 1:200 dilution of aCy3-conjugated anti-rabbit secondary antibody in PBS containing 1% BSAfor 1 h at RT. Finally, cells were washed with PBS, fixed with PBScontaining 4% PFA, mounted, and visualized in an optical fluorescencemicroscope.

Rescue of Recombinant AAV from Cells Transduced by AAVP Particles. Human293 cells were infected with RGD-4C AAVP-GFPneo or RGD-4C phage-GFPneo.Four days after infection, cells were transfected with an AAV rep- andcap-expressing plasmid (pXX2) (Xiao et al., 1998) and superinfected withwild-type adenovirus type 5 (Ad). Thus, the AAV rep and cap genes weresupplied by transfection, and adenovirus helper functions were providedby superinfection. Cells were harvested 72 h post-adenoviral infectionand supernatants were then used to infect new 293 cells. GFP expressionwas analyzed by using FACS 48 h later. In this assay, a functionalrecombinant AAV was generated from cells transduced with the RGD 4CAAVP-GFP chimera only, but not from cells transduced with thenon-chimeric phage-GFP or several controls. Similar results were alsoobtained with all the RGD-4C AAVP clones but with none of the phageclones.

Generation of Clonal Mammalian Cell Lines. Human 293 cells were infectedwith RGD-4C AAVP-GFPneo or RGD-4C phage-GFPneo (at 10⁶ TU per cell ineach case). Single-clones (n=9 per group) were isolated under G418selection and analyzed for GFP expression by FACS at 12 weeks afterselection. Stable clones were termed phage clones #1-9 for phage-GFPneoand AAVP clones #1-9 for AAVP-GFPneo.

Tumor Models. Animal experimentation was reviewed and approved by theInstitutional Animal Research Committee. Tumor-bearing mice wereestablished as described (Pasqualini et al., 1997; Arap et al., 1998;Ellerby et al., 1999; Hajitou et al., 2001; Arap et al., 2004; Marchibet al., 2004). Mice were anesthetized by intraperitoneal administrationof AvertinB or by gas (2% isoflurane and 98% oxygen) inhalation.Targeted constructs or controls were intravenously administered. Tumorcells were trypsinized, counted, centrifuged, and re-suspended in serumfree medium. A total of 10⁶ cells from Kaposi sarcoma (KS1767), bladdercarcinoma (UC3) or prostate carcinoma (DU145) lines were implantedsubcutaneously into 6 week-old immunodeficient nude mice. The EF43-FGF4mouse mammary tumor cells (5×10⁴) were implanted subcutaneously into 6week-old female BALBIc immunocompetent mice. Tumor volumes werecalculated as described (Pasqualini et al., 1997; Arap et al., 1998;Ellerby et al., 1999; Hajitou et al., 2001; Arap et al., 2004; Marchibet al., 2004) and expressed as mean tumor volume*standard deviation(SD). When tumors reached a volume of −50 mm² (deemed small) or −150 mm²(deemed large) DU145-derived xenografts (day 0), tumor-bearing micereceived a single intravenous dose of RGD-4C AAVP-HSVtk, or controls.GVC treatment (80 mg/kg per day, intraperitoneal) started two days laterin cohorts of size-matched tumor-bearing mice.

Molecular-genetic Imaging in Tumor-bearing Mice. For non-invasivemolecular imaging, we used a model of prostate cancer based on the humancell line DU145 in which male nude mice bearing tumor xenografts in thesubcutaneous area of the right shoulder were used. To image the fireflyLuc gene expression, tumor-bearing mice received a single-dose (1 50mg/kg) of the substrate D-luciferin (Xenogen) by intraperitonealadministration. Photonic emission was imaged by using the In VivoImaging System 200 (IVIS200; Xenogen, CA) after tail vein administrationof targeted RGD-4C AAVP carrying the Luc gene or controls (non-targetedAAVP-Luc, or scrambled RGD-4C AAVPLuc). Imaging parameters: imageacquisition time, 1 min; binning, 2; no filter; flstop, 1; field ofview, 10 cm. Regions of interest (ROI) were defined manually over thetumors for measuring signal intensities, expressed asphotons/sec/cm2/sr.

While BLI can assess transgene-expressing cell viability in experimentalsystems, it is not clinically applicable. Thus, to assess the viabilityof the established tumor xenografts, mice were imaged with a microPETscanner (Concorde Microsystems, TN) at 2 h post intravenousadministration of [¹⁸F]-FDG 100 μCi/mouse. [¹⁸F]-FDG was obtainedcommercially (PETNet, Houston, Tex.). To image HSVtk gene expression,PET imaging was performed at 1-2 h after intravenous administration ofthe radiolabeled nucleoside analog [¹⁸F]-FEAU. PET imaging was performedon a microPET R4 (Concorde Microsystems, Inc.), equipped with acomputer-controlled positioning bed, has a 10.8-cm transaxial and 8-cmaxial field of view (FOV), it has no septa and operates exclusively in3-dimensional list mode. Fully 3-dimensional list mode data werecollected using an energy window of 350-750 keV and a time window of 6ns. All raw data were first sorted into 3-dimensional sinograms,followed by Fourier rebinning and OSEM image reconstruction using ASIPROVM software (Concord Microsystems, TN). Image pixel size wasapproximately 1 mm transaxially with a 1.2 mm slice thickness.

Radiolabeled [¹⁸F]-FEAU was synthesized to radiochemical purity greaterthan 99% by using 5-ethyluracil-2,5-bis-trimethylsilyl ether as thepyrimidine base for condensation with1-bromo-2-deoxy-2-[¹⁸F]fluoro-3,5-di-O-benzoyl-α-D-arabinofuranose, asoriginally described by Alauddin et al. (2003). To quantitate the[¹⁸F]-FEAU or [¹⁸F]-FDG-derived radioactivity concentration in tumorsand other organs and tissues, regions of interest were drawn on imagesand the measured values converted from nCi/mm² into % injected dose pergram (% ID/g; Tjuvajev et al., 1998). Repetitive [¹⁸F]-FEAU PET imagingwas performed at days 3, 5, 10, and 16 post administration of targetedAAVP carrying the HSVtk gene or control non targeted AAVP, as describedabove; GCV treatment was administered between days 11 and 19. Of note,PET imaging at day 16 was performed at 24 h after GCV dosing to allowfor sufficient elimination of GCV, which would otherwise compete withFEAU for phosphorylation by the HSVtk enzyme. PET imaging with [¹⁸F]-FDGwas repeated at day 17 after AAVP administration to assess the viabilityof residual tumor, if any.

Immunohistochemistry. Anesthetized mice were killed and perfused withPBS containing 4% PFA. Tumor vascularization was assessed on frozensections by using a rat anti-mouse CD31 antibody (BD Biosciences).Apoptosis analysis was performed on paraffin-embedded sections with aTUNEL kit (Promega). For phage immunodetection in tissues, paraffinsections were incubated with a rabbit anti-phage primary antibody(Sigma) followed by a peroxidase-conjugated anti-rabbit secondaryantibody (Dako). Slides were developed with the substrate-chromogen3,3′-diaminobenzidine and counterstained with hematoxylin. For GFPimmunostaining, organs and tumors were fixed for 2 h in PBS containing2% PFA and equilibrated for 48 h in PBS containing 15% sucrose.Cryosections were post-fixed in PBS containing 4% PFA for 20 min andblocked with 5% goat serum in PBS containing 1% BSA and 0.1% TritonX-100 (PBS-T). Next, tissue sections were incubated with a rabbitaffinity purified GFP antibody (Molecular Probes) in 2% goat serum and1% BSA. Sections were then stained with the secondary antibodyAlexaFluor 488 conjugated goat anti-rabbit (Molecular Probes) in PBS-Tand 1% BSA. αv integrin immunostainings were performed on acetone fixedfrozen sections of tumors removed from PBS-perfused animals. Sectionswere incubated for 1 h with the primary rat anti-integrin αv monoclonalantibody (Chemicon), followed by the secondary Cy3 conjugated goatanti-rat antibody (Jackson ImmunoResearch).

Ligand-directed Particles Are Functional in Mammalian Cells. A targetedchimeric virus comprising of recombinant AAV and an fd-tet phage clonedisplaying the double-cyclic peptide CDCRGDCFC (SEQ ID NO:2) (termedRGD-4C phage (Pasqualini et al., 1997; Arap et al., 1998) wasconstructed. The RGD-4C peptide binds to αv integrins, a cell surfacereceptor over-expressed in both tumor cells and in neo-angiogenicendothelium of tumor blood vessels (Brooks et al., 1994; Pasqualini etal., 1997; Arap et al., 1998; Sipkins et al., 1998; Ellerby et al.,1999; Hood et al., 2002). To obtain chimeric viruses (further referredto as AAV/phage; AAVP), the inventors inserted an eukaryotic genecassette from recombinant AAV in an intergenomic region of RGD-4C phage(RGD-4C AAVP), insertless phage (non-targeted AAVP), or phage displayingcontrol peptides, such as scrambled RGD-4C AAVP or D to E mutant (termedRGE-4C) AAVP, and packaged it with the phage DNA into the phage capsid(FIG. 7). In order to show that the cis-elements of the resulting αvintegrin targeted chimeric virus remain functional, the inventorsevaluated the ligand properties of the RGD-4C peptide and the rescuingproperties of the inverted terminal repeats (ITRs) in the context ofAAVP. First, to evaluate peptide specificity, it was shown that RGD-4CAAVP binds to mammalian cells expressing αv integrins, in contrast tothe non-targeted AAVP or AAVP displaying negative control peptides suchas RGE-4C or various scrambled versions of the RGD-4C sequence (FIG.1A), which neither bind to nor infect mammalian cells. It was alsodemonstrated that RGD-4C AAVP carrying reporter genes can mediateligand-directed internalization (FIG. 1B) and transduction of mammaliancells (FIG. 1C) relative to controls. For cell internalizationexperiments, negative controls included non-targeted AAVP, variousRGD-4C scrambled AAVP, or RGE-4C AAVP (FIG. 1B); for cell transductionexperiments, non-targeted AAVP (FIG. 1C), scrambled RGD-4C AAVP, orRGE-4C AAVP served as negative controls. Consistent with these results,the inventors have previously demonstrated that the synthetic RGD-4Cpeptide specifically inhibits cell binding and internalization ofvarious targeted RGD-4C phage based constructs (Giordano et al., 2001;Chen et al., 2004, and unpublished results). Finally, it was shown thatmammalian cell transduction can also be specifically competed by thesynthetic RGD-4C peptide relative to negative controls such asnon-targeted AAVP (FIG. 1D), Scrambled RGD-4C, or RGE-4C. To rule outthe possibility that some of these results (FIGS. 1A and 1B) couldrepresent an artifact resulting from selective failure of the glycine(low pH) wash step to remove AAVP from cell membranes, temperature(ice-cold) control experiments were performed (Giordano et al., 2001) inwhich cell binding was observed but not internalization mediated byRGD-4C AAVP.

Next, to evaluate whether the ITRs are still functional in the AAVPparticle we performed rescue experiments. We show that functionalrecombinant AAV particles are generated from mammalian cells transducedwith the RGD 4C AAVP only, but not from the cells transduced withnegative control constructs (FIG. 1E). These data establish that thegenetic chimerization resulting in an RGD-4C AAVP particle does notfundamentally alter (i) the peptide targeting properties of theligand-directed RGD-4C phage or (ii) the ability to rescue recombinantAAV particles from mammalian cells transduced by RGD-4C AAVP. Aside-by-side time course of a reporter transgene expression revealedthat RGD-4C AAVP transduction was detectable for much longer compared tothat of the RGD-4C phage (Table 4).

TABLE 4 Transgene expression in vitro. Post-infection day # 0 5 10 15 2030 35 40 45 50 55 60 RGD-4C-phage − ++ + − − − − − − − − − RGD-4C- − ++++ ++ ++ ++ ++ ++ ++ + + + AAVP Two independent observers acored GFPexpression semi-quantitatively in hiplicate wells of 293 cells per timepoint.

Molecular Mechanisms of Transgene Expression. To gain an insight intothe molecular mechanisms of transgene expression mediated by AAVPparticles, the inventors investigated the fate of the transduced genomein mammalian cells. First, stably transduced cell lines were generatedby using GFPneo-expressing AAVP to allow for the selection of individualtransduced cell clones. Either RGD-4C AAVP-GFPneo or RGD-4C phage-GFPneolacking AAV ITRs were used to transduce human 293 cells expressing αvintegrins (Nakamura et al., 2002), and clones were isolated under G418selection. Rescue experiments showed functional recombinant AAV-GFPneogenerated from all the RGD-4C AAVP-GFPneo 293 cell clones, but nonegenerated from the RGD-4C phage-GFPneo clones, thus confirming that thechimeric individual clones contain functional ITRs. Although cellstransduced with the non-chimeric RGD-4C phage-GFPneo retainedG418-resistance, GFP expression was generally weaker than that of theRGD-4C AAVP-GFPneo clones (FIG. 2A). The inventors then set out todetermine the fate of the GFPneo transgene cassette in stably transducedclones by a comprehensive restriction enzyme digestion of genomic DNAfollowed by Southern blotting and polymerase chain reaction (PCR)-basedanalysis (FIG. 2, FIG. 8, and Table 5). To prove persistence of thetransgene cassette, genomic DNA was digested with AflII and XhoI todetect the release of full-length transgene cassettes prior to theanalysis. Such release was observed in 100% of the clones transduced byRGD-4C AAVP (n=9 of 9 clones), compared to only in 33% of the clones(n=3 of 9 clones) transduced by the non-chimeric RGD-4C phage construct(FIG. 2B). Another restriction digest of genomic DNA was designed todetect potential concatemeric forms of the transgene cassette (Lieber etal., 1999; Hsiao et al., 2001). The presence of head-to-tail concatemersof the transgene cassette was detected in 67% of the clones transducedby RGD-4C AAVP (n=6 of 9 clones) while no such concatemers were detectedin clones transduced by the non-chimeric RGD-4C phage construct (FIG.2C). To identify possible additional concatemeric forms of the transgenecassette, multiplex PCR was performed by using primers flanking the 5′and 3′ ends of the constructs. Again, no concatemers were found inclones transduced by non-chimeric RGD-4C phage construct, whereas 100%of clones transduced by RGD-4C AAVP contained concatemeric forms (n=9 of9 clones), all of them found in head-to-tail orientation (FIG. 8B).Moreover, topo cloning of smaller PCR products revealed the head-to-tailorientation of the transgene cassette with ITR deletions (FIG. 8C) (Yanget al., 1997). Finally, although the large PCR products could not besequenced, their sizes suggest the presence of concatemers with intactITRs at the junction site. An individual analysis of DNA for each singleclone is also detailed (Table 5). Not wishing to be bound by theory ormechanism, these data suggest that AAVP may bestow an advantage in geneexpression by means of an altered fate of the transgene cassette throughmaintenance of the entire mammalian transgene cassette, betterpersistence of episomal DNA, formation of concatemers of the transgenecassette, or perhaps by a combination of these non-mutually exclusivemechanisms. These observations are consistent with recent developmentsin the understanding of AAV (McCarty et al., 2004).

TABLE 5 Fate of the vector DNA in 293 cell clones stably transduced byRGD-4C AAVP-GFneo or RGD-4C phage-GFneo. RGD-4C phage or RGD-4C AAVPContains (denomination Contains Contains concatemeric of stablyintegrated episomal forms of Transgene transduced forms of forms oftransgene cassette clones) construct construct cassette preservedComments and Interpretation Phage 1 Yes No No No Phage 2 Yes Yes No YesEpisomal form of full-length phage (episomal) vector Phage 3 Yes No NoYes Phage 4 Yes No No No Phage 5 Yes No No No Phage 6 Yes Yes No YesEpisomal form of full-length phage (episomal) vector Phage 7 Yes No NoNo Phage 8 Yes No No No Phage 9 Yes No No No AAVP 1 Yes Yes Yes YesNon-concatemeric episomal forms of full-length AAVP vector detected.Concatemeric integrated form with deleted Xhol Site. AAVP 2 Yes No YesYes Concatemeric form is head-to-tail with deleted ITRs at the junctionsite. ITRs flanking the concatemer and adjacent AAVP sequences arepreserved. AAVP 3 Yes Yes Yes Yes Non-concatemeric episomal form offull-length AAVP vector. At least one integrated form contains head-to-tail concatemers and ITRs flanking the concatemer as well as theadjacent AAVP sequences are preserved. At least one integrated formcontains head-to-tail concatemers with deleted ITRs at the junctionsite. AAVP 4 Yes Yes Yes Yes Non-concatemeric episomal form offull-length AAVP vector. At least one integrated form contains head-to-tail concatemers and ITRs flanking the concatemer as well as theadjacent AAVP sequences are preserved. At least one integrated formcontains head-to-tail concatemers with deleted ITRs at the junctionsite. AAVP 5 Yes Yes Yes Yes Non-concatemeric episomal form offull-length AAVP vector. At least one integrated form contains head-to-tail concatemers and ITRs flanking the concatemer as well as theadjacent AAVP sequences are preserved. At least one integrated formcontains head-to-tail concatemers with deleted ITRs at the junctionsite. AAVP 6 Yes No Yes Yes Concatemeric are head-to-tail with deletedITRs and Xhol sites. ITRs flanking the (concatemer) transgene cassetteas well as the adjacent AAVP sequences are preserved. AAVP 7 Yes No YesYes Concatemers are head-to-tail with preserved ITRs. Additionalconcatemers with deleted ITRs. AAVP 8 Yes No Yes Yes At least oneintegrated form contains head-to-tail concatemers and ITRs flanking theconcatemer as well as the adjacent AAVP sequences are preserved. Atleast one integrated form contains head- to-tail concatemers withdeleted ITRs at the junction site. AAVP 9 Yes Yes Yes YesNon-concatemeric episomal form of full-length AAVP vector. At least oneintegrated form contains head- to-tail concatemers and ITRs flanking theconcatemer as well as the adjacent AAVP sequences are preserved. Atleast one integrated form contains head-to-tail concatemers with deletedITRs at the junction site.

Tumor Targeting In Vivo and Molecular-Genetic Imaging. Afterdemonstrating that the central elements of the targeted chimeric viralparticle (i.e., the RGD-4C peptide and the AAV ITRs) are intact andfunctional and after elucidating the molecular mechanisms ofAAVP-mediated gene expression in mammalian cells, the specificity andefficacy of gene delivery into tumors after systemic administration ofAAVP was evaluated. As an initial preclinical model, nude mice bearingsubcutaneous tumor xenografts derived from human Kaposi sarcoma KS 1767cells were used (Arap et al., 1998; Ellerby et al., 1999). First, toverify that the viral construct targets to KS1767-derived xenografts inmice, either RGD-4C AAVP or one of several negative controls(non-targeted AAVP, scrambled RGD-4C AAVP, or RGE-4C AAVP) wereadministered intravenously. After a 3-5 min circulation time, a stronganti-AAVP staining in tumor vasculature was observed in mice thatreceived RGD-4C AAVP but not in control mice (FIG. 3A). An RGD-4C AAVPvariant encoding the green fluorescent protein (GFP) gene was used as areporter to determine, by using in situ immunofluorescence microscopicimaging, whether this vector (RGD-4C AAVP-GFP) can transduce KS1767-derived xenografts. Immunostaining against GFP in tumors and indifferent organs were performed seven days after systemic administrationof either RGD-4C AAVP-GFP or negative control constructs intotumor-bearing mice. Immunofluorescence revealed GFP expression largelyin tumor blood vessels and surrounding tumor cells in mice that receivedRGD4C AAVP-GFP. In contrast, no GFP staining was detected in tumors fromcontrol mice that received non targeted, scrambled, or mutant AAVP-GFP(FIG. 3B). This staining pattern suggests that ligand-directedtransduction is mediated by targeting of αv integrins in the vascularendothelium of tumors (Brooks et al., 1994; Pasqualini et al., 1997;Arap et al., 1998; Sipkins et al., 1998; Ellerby et al., 1999; Giordanoet al., 2001; Hood et al., 2002; Chen et al., 2004). Consistently,several non-target control organs (brain, liver, pancreas and kidney)lacked tissue expression of GFP (FIG. 9). Together, these resultsindicate that RGD-4C AAVP particles can specifically target tumorxenografts by a ligand-directed mechanism and transduce them aftersystemic administration in vivo.

Next, inventors assessed the efficacy of preclinical bioluminescenceimaging (BLI) and clinically applicable molecular-genetic PET imagingwith [¹⁸F]-FEAU for non-invasive monitoring of temporal dynamics andspatial heterogeneity of the firefly luciferase (Luc) and of the HSVtkreporter gene expression, respectively, in living tumor-bearing micefollowing systemic administration of the RGD-4C AAVP. Thesemolecular-genetic imaging studies were conducted in a preclinical modelof human prostate cancer since this particularly prevalent tumor remainsa challenge to properly image in patients. The inventors first used astandard experimental setup for in vivo imaging of the fireflyLuciferase (Luc) transgene reporters in tumor-bearing mice (FIG. 4A).The inventors selected BLI of Luc expression, because it is a verysensitive method for reporter gene imaging in mice and has virtually nonon-specific background activity in the images (Gross and Piwnica-Worms,2005a; Gelovani and Blasberg, 2003; Uhrbom et al., 2004; Walensky etal., 2004). A very tumor-specific expression of Luc was observed inDU145 tumors in mice receiving RGD-4C AAVP-Luc. In contrast,tumor-associated bioluminescence signals could not be observed incontrol mice receiving the non-targeted AAVP-Luc or scrambled RGD-4CAAVP-Luc. With all types of AAVP vectors (non-targeted AAVP-Luc,scrambled RGD-4C AAVP-Luc, RGE-4C AAVP-Luc or RGD-4C AAVP-Luc) nobioluminescence was observed in normal organs such as liver, spleen, orkidneys. These data confirm the tumor-specificity of RGD-4CAAVP-mediated targeting and transgene expression observed withimmunofluorescence microscopy imaging studies with RGD-4C AAVP-GFP.Consistently with previous results presented here (FIG. 9), the kineticsof distribution suggests that despite the non-specific hepatic clearanceof phage particles (Geier et al., 1973; Pasqualini et al., 1997; Arap etal., 1998; Barbas et al., 2001), such phenomenon does not result in anundesirable gene transduction of the liver. These observations are insharp contrast with the well-documented non-specific transduction ofnormal organs (such as liver) by the mammalian viral gene deliveryvectors (Shayakhmetov et al., 2005). By using BLI in vivo, Luc reportertransgene expression within tumors was clearly detectable at day 3 afterAAVP administration and increased gradually to reach maximal levels byday 10. Repetitive 2-dimensional BLI of Luc reporter gene expression wasperformed every other day and provided an initial cost effectivestrategy to study the specificity, temporal dynamics, and spatialheterogeneity of reporter transgene expression mediated by AAVP.However, because BLI of Luc reporter gene expression is not clinicallyapplicable, the inventors next introduced into the AAVP vector the HSVtkgene, which can serve both as a suicide gene (when combined withgancyclovir; GCV) and as a reporter transgene for clinically applicablePET imaging with HSVtk-specific radiolabeled nucleoside analogues (e.g.,[¹²⁴I]-FAIU, [¹⁸F]-FHBG, and [¹⁸F]-FEAU).

Previous studies established that PET imaging of HSVtk expressionprovides the ability to define the location, magnitude, and duration oftransgene expression (Tjuvajev et al., 1998; Tjuvajev et al., 1999; Rayet al., 2001; Massoud and Gambhir, 2003). It has also been previouslydetermined that the magnitude of accumulated radiolabeled tracers inHSVtk-transduced cell lines and tumors in vivo correlates with the levelof HSVtk expression (Blasberg and Tjuvajev, 2003; Gross andPiwnica-Worms, 2005a; Tai and Laforest, 2005). In the studies presentedhere, the inventors selected, synthesized and used the radiolabelednucleoside analogue2′-[¹⁸F]-fluoro-2′-deoxy-1-β-D-arabino-furanosyl-5-ethyl-uracil([¹⁸F]-FEAU), which is a better radiolabeled substrate for the HSVtkenzyme than other nucleoside analogues, especially from pharmacokineticconsiderations (a very low background activity in all normal organs andtissues) (Kang et al., 2005). By using repetitive PET imaging with[¹⁸F]-FEAU (on days 0, 3, 5, 10, and 16), the inventors have visualizedand quantitated the temporal dynamics and spatial heterogeneity of HSVtkgene expression after a single systemic administration of RGD-4CAAVP-HSVtk or non-targeted AAVP-HSVtk in DU145-derived tumor xenograftsand other organs and tissues in nude mice (FIG. 4B). Tumor xenograftsizes (approximately 150 mm²) before, as well as tumor growth ratesafter administration of either RGD4C AAVP-HSVtk or non-targetedAAVP-HSVtk were similar in both cohorts of mice (FIG. 4C). PET imagingwith [¹⁸F]-FEAU revealed a gradual increase in the level of HSVtktransgene expression in tumors (increase in % administered intravenousdose per gram) during the initial five days after administration ofRGD-4C AAVP-HSVtk, followed by gradual stabilization of HSVtk expressionlevels towards day 10 post vector administration. In contrast, incontrol tumor-bearing mice receiving nontargeted AAVP-HSVtk, only aminor increase in tumor accumulation of [¹⁸F]-FEAU was observed at day3, which rapidly decreased to background level (FIG. 4D). Consistentwith preceding BLI experiments, no [¹⁸F]-FEAU PET detectable HSVtkexpression was observed in non-target organs or tissues (FIG. 4B).Indeed, low-level heterogeneous activity in the PET images representsnormal background activity, which was intentionally intensified inimages to demonstrate that no truncation of low levels of radioactivitywas made to artificially “improve” the specificity of HSVtk expressionin tumors versus non-target tissues. When tumors grew to reliablypalpable sizes (approximately 350-400 mm²), and a plateau of HSVtkexpression was achieved in tumors, treatment with GCV was initiated inall cohorts of animals (FIG. 4C). PET imaging with[¹⁸F]-fluorodeoxyglucos ([¹⁸F]-FDG) served to monitor glucose metabolismand GCV-induced changes in tumor viability. Two days before initiationof GCV therapy (day 9 post vector administration), the DU145 tumors inboth groups of mice were viable and actively accumulated [¹⁸F]-FDG (FIG.4E). After GCV therapy, the volume of tumors in mice that receivedRGD-4C AAVP-HSVtk was significantly smaller than in mice that receivednon-targeted AAVP-HSVtk (p<0.05; FIG. 4C). Moreover, tumor xenograftswere also metabolically suppressed as evidenced by a decrease inaccumulation of [¹⁸F]-FDG (FIG. 4E). The levels of HSVtk expression intumors of mice administered with RGD-4C AAVP-HSVtk were alsosignificantly decreased after GCV therapy, as evidenced by a sharpdecrease in [¹⁸F]-FEAU accumulation in PET images (FIG. 4D), which wereobtained 24 h after the last GCV dose (to avoid competition with FEAU).These studies confirm the specificity of tumor targeting by RGD-4C AAVPand demonstrate that the level of HSVtk transgene expression isadequately high for effective prodrug activation of GCV.

In order to evaluate efficacy horizontally in other preclinical models,we assembled a panel of tumor cell lines from different species andhistological origins and generated tumors in immunosuppressed orimmunocompetent mice. Cohorts of Kaposi sarcoma (KS 1767)-derivedtumor-bearing mice received systemically a single intravenous dose ofeither the RGD-4C AAVP-HSVtk or non-targeted AAVP-HSVtk (control),followed by GCV treatment in all groups. Marked tumor growth suppressionwas observed in tumor-bearing mice receiving RGD-4C AAVP-HSVtk, ascompared to mice treated with vehicle or mice that received non-targetedAAVP (FIG. 5A). Similar tumor growth suppressive effects were observedin UC3-derived bladder carcinomas (FIG. 5B) and DU145-derived prostatecarcinomas (FIG. 5C) in nude mice, even if larger tumor xenografts weretreated (FIG. 5D) and consistent with the imaging results presented(FIG. 4). To rule out the possibility that the observed anti-tumoreffects were either species-specific or xenograft-specific, we sought toanalyze the efficacy of the RGD-4C AAVP-HSVtk on a standard mouse tumormodel. We chose an isogenic tumor in which EF43-FGF4 mouse mammary cellsare administered subcutaneously to induce rapid growth of highlyvascularized tumors in immunocompetent mice (Hajitou et al., 2001).First, we show ligand-directed homing of RGD-4C AAVP to EF43FGF4-derived tumors by anti-phage immunostaining (FIG. 10). EitherRGD-4C AAVP-GFP or non-targeted AAVP-GFP was administered intravenouslyto mice bearing isogenic mammary tumors for a 3-5 min circulation time.Unlike the non-targeted AAVP-GFP, RGD-4C AAVP GFP produced a stronganti-phage staining in tumors (FIG. 10A). Next, we performedimmunofluorescence with an anti-GFP antibody at seven days afterintravenous administration to reveal strong GFP expression in the tumorsin mice that received RGD-4C AAVP-GFP; in contrast, no GFP staining wasdetected in tumors from mice that received non-targeted AAVPGFP;consistently, an anti-av integrin antibody detected strong expression inEF43-FGF4 tumors (FIG. 10B). Again, a single systemic dose of RGD-4CAAVP-HSVtk followed by GCV markedly inhibited the growth of EF43-FGF-4tumors (FIGS. 5E-G and FIG. 6). Moreover, when tumors grew back aftertermination of therapy, repeated administrations of RGD-4C AAVP-HSVtkagain inhibited EF43-FGF4 tumor growth and improved survival of tumorbearing mice (FIG. 5F). Phage-based particles are known to beimmunogenic but this feature can be modulated through targeting itself(Trepel et al., 2001). In fact, RGD-4C AAVP-HSVtkplus GCV remainedsurprisingly effective on phage-vaccinated immunocompetent mice despitevery high titers of circulating anti-phage IgG (FIG. 5G). In selectiveexperiments, a comprehensive panel of negative experimental controlsincluding vehicle alone, vehicle plus GCV, non-targeted AAVP,non-targeted AAVP plus GCV, targeted RGD-4C AAVP, targeted RGD-4CAAVP-GFP, and targeted RGD-4C AAVP-GFP plus GCV (mock transduction) wereused (FIG. 6A).

To check for post-treatment effects, the inventors obtained detailedhistopathological analysis of EF43 FGF4 tumors recovered seven daysafter therapy. Extensive tumor destruction caused by the single systemicdose of RGD-4C AAVP-HSVtk plus GCV was noted. Specifically, hematoxylinand eosin (H&E) staining revealed uniform destruction of the centralarea of the tumor and only a small viable outer rim; in contrast,non-targeted AAVP-HSVtk had no such effect (FIG. 6B). Staining with ananti-CD31 antibody confirmed both disrupted tumor blood vessels withinthe tumor central region and preserved vasculature towards the outerrim, whereas no damage was observed in the tumors treated withnon-targeted AAVP-HSVtk (FIG. 6B). The inventors also evaluated thetumors for terminal deoxynucleotidyl transferase-mediated dUTP biotinnick end-labeling (TUNEL) staining, which marks apoptotic cells, becausethe HSVtk/GCV strategy is associated with apoptotic death of cells(Hamel et al., 1996). In tumors treated with RGD-4C AAVP-HSVtk and GCV,TUNEL staining detected apoptosis in the tumor central region but notwithin the outer rim while no apoptosis was observed in tumors from micethat received non-targeted AAVP-HSVtk chimera (FIG. 6B). Control organsremoved from tumor-bearing mice treated by the same experimentalprotocol revealed no histopathologic abnormalities (FIG. 11). Together,these results show that a single systemic dose of RGD-4C AAVP-HSVtk plusGCV maintenance can suppress tumor growth.

The relative contribution of targeting each tumor compartment (i.e.,tumor cells versus tumor vascular endothelium and/or stroma) will dependon the ligand-receptor system and on the model(s) used. For instance,the expression of the membrane target (i.e., αv integrins) in tumorcells will vary from low (EF43 FGF4) to strong (KS1767). Moreover, asidefrom the specific optimal dose for transduction of each model (andapplication) used, there is also an optimal time to examine transgeneexpression. In other words, the stoichiometry of reporter geneexpression depends not only from levels and patterns of reporterexpression in individual cells, but also from the relative number ofproliferating transgene-expressing cells versus dyingtransgene-expressing cells. Thus, while the inventors used fixedparameters for these examples, further determination of targeted AAVPoptimal doses and timeframes on a case-by-case basis still apply.

The inventors contemplate that a broad range of currently intractablebiological questions well beyond molecular oncology can be addressedusing targeted AAVP, especially in combination with differentpre-clinical and clinical molecular-genetic imaging settings. Forexample, the systemic ligand-directed delivery of constructs with tissueand/or disease-specific promoters (instead of the CMV promoter) totarget sites will allow monitoring expression of their correspondingnative genes in vivo; such promoter-driven transcription of reporteractivity will allow the study of cell trafficking and engraftment.Several non-invasive imaging applications can be employed such asexperimental monitoring of substrate-specific degradation,protein-protein interactions and other molecular events via reportertransactivation, complementation, or reconstitution strategies (Luker etal., 2004; De and Gambhir, 2005; Gross and Piwnica-Worms 2005b) in cellsand in whole animals. The inventors have recently described networks ofgold nanoparticles and bacteriophage as biological sensors and celltargeting agents (Souza et al., 2006), such technology can be combinedwith ligand-directed AAVP to further improve molecular-genetic imaging.On another note, AAVP itself may provide suitable reagents to study themechanistic role of ITR structures in transgene persistence andchromosomal integration since (in contrast to AAV vectors) phage-basedconstructs with no ITRs can serve as negative experimental controls.Thus, many other systemic targeting and molecular imaging applicationsin tandem is possible in a relatively short timeframe with this novelplatform and its derived tools.

Example 2 Use of AAVP to Target TNF-α to Tumor Vasculature

A. Methods

Cell Culture. Human umbilical vein endothelial cells (HUVEC) wereobtained from Cambrex (Walkersville, Md.) and cultured in EndothelialCell Growth Medium-2 as described previously (Tandle et al., 2005). Allexperiments were conducted with HUVEC in passage 3-5. M21 human melanomacells were grown in RPMI 1640 medium containing 10% serum, 2 mMglutamine, 100 u/ml penicillin, 100 μg/ml streptomycin, 100 μg/mlgentamicin and fungizone. Pmel cells were grown in DMEM mediumcontaining 10% serum, 2 mM glutamine, 100 u/ml penicillin, 100 μg/mlstreptomycin, 100 μg/ml gentamicin and fungizone.

Construction of a Targeted AAVP Expressing TNF-α/EMAP-II. The generaldesign and construction of the AAVP backbone is described in Hajitou etal. (2006). An AAVP construct expressing TNF-α was created in two steps.First, a 880 bp NotI/HindIII fragment from pG1SiTNF was digested andligated into a pAAV-eGFP/NotI/HindIII vector replacing GFP genesequences (Hwu et al., 1993). In the second step, fMCS/RGDMCS andAAV-TNF were digested by PvuII. Then AAV-TNF-α with inverted terminalrepeats (ITRs) were religated into the fMCS-/RGDMCS PvuII site to obtainan AAVP vector. Thus, pfdTNF-α is a non-targeted vector, whereas pRGDTNFis a targeted vector with binding affinity to cell surface αv integrinreceptors.

An AAVP construct expressing EMAP-II was created in three steps. ThepET-20b plasmid, expressing mature EMAP-II was a generous gift from PaulSchimmel at The Scripps Research Institute, La Jolla, Calif. In thefirst step, EMAP-II sequences were amplified from pET-20bEMAP-II, using3 primers to incorporate restriction enzyme sites and the secretablesignal sequence in the PCR product. Primer 1 is a 132 bp forward primerwith NotI restriction enzyme site at the 5′ end followed by a signalsequence (designed from pSecTag2 vector, Invitrogen, Carlsbad, Calif.)for extracellular secretion of gene product and EMAP-II sequences.Primer 2 is a shorter version of primer 1 to facilitate amplification ofthe PCR product. Primer 3 is a reverse primer with a HindIII restrictionenzyme site at 3′ end. The PCR amplification generated a 667 bp productwith NotI and HindIII enzyme sites and signal sequence. In the secondstep, the EMAP-II PCR product was cloned into the pCR11-TOPO cloningvector (Invitrogen, Carlsbad, Calif.). The resultant clones weresequenced, and then the 667 bp NotI/HindIII fragment was ligated into apAAV-eGFP/NotI/HindIII vector as explained earlier. In the third step,fMCS/RGDMCS and AAV-EMAP-II were digested by PvuII and ligated to obtainan AAVP vector. Thus, pfdEMAP-II is a non-targeted vector, whereaspRGDEMAP-II is a targeted vector with binding affinity to cell surfaceαv integrin receptors. Primer 1: 5′ATTTGCGGCCGCTTTACCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACGCGGCCCAGCCGGCCAGGCGCGCCGTAATGTCTAAGCCAATAGATGTT 3′ (SEQ ID NO:4); Primer 2:5′ATTTGCGGCCGCTTTACCACCATGG 3′ (SEQ ID NO:5); Primer 3: 5′CCCAAGCTTGGGTTATTTGATTCCACTGTTGC 3′ (SEQ ID NO:6).

AAVP Particle Purification. To obtain non-targeted and targeted AAVPparticles, DNA was electroporated into MC1061 E. coli. Cells and virusparticles were purified from the culture supernatant. Large scale AAVPparticles were purified from permissive k91Kan cells. In order todetermine the number of bacterial transducing units (TU), k91 cells wereinfected with serial dilutions of phage particles and plated onLuria-Bertani agar plates containing tetracycline and kanamycin TU wasthen determined by counting the number of bacterial colonies.

In Vitro Phage Internalization Assay. M21 cells were grown overnight in6-well tissue culture plates. To study vector internalization, cellswere washed with the media and then infected with viral particles at 37°C. for 3 hrs. After incubation, plates were placed on ice for 5 min inorder to stop viral internalization. Unbound particles were removed byextensive washing of cells in Hank's balanced salt solution (HBSS).Extracellular viral particles were inactivated by treatment withSubtilisin (3 mg/ml subtilisin, 20 mM Tris pH 7.5, 2 mM EDTA pH 8.0 inHBSS with no calcium and no magnesium) for 1 hr on ice (Ivanenkov etal., 1999). Then, cells were detached with gentle pipetting andsubtilisin was inactivated with 2 mM EDTA on ice for 15 min. Cells werelysed by using lysis buffer (10 mM Tris pH 7.5, 2 mM EDTA pH 8.0 and 2%sodium orthovanidate) to release the internalized AAVP particles. Theinternalized AAVP concentration was then determined as TU as describedabove.

Immunofluorescence (IF) Assay. IF was used to observe internalized viralparticles in M21 cells. Briefly, cells grown on 8-well Lab-Tek chamberglass slides (Nunc, Rochester, N.Y.) were infected with AAVP particlesby using DMEM containing 10% serum at 37° C. for 16 hrs. Afterinfection, cells were washed with PBS, chambers were removed and cellswere fixed in 3.7% formaldehyde for 10 min. Cells were permeabilized by0.1% saponin (Sigma, St. Louis, Mo.) in PBS, and blocked with blockingbuffer (PBS containing 1% BSA, 0.025% sodium azide, and 0.1% saponin)for 15 min. After washing with permeabilization buffer, cells wereincubated with a mouse anti-bacteriophage antibody for 1 hr, followed bya FITC-conjugated anti-mouse IgG antibody for 1 hr. Gaskets weredetached and cells were mounted using Antifade (MP Biomedicals, Solon,Ohio) and examined under a Zeiss Axiovert fluorescent microscope.

Gene Expression by Phage. M21 cells were infected with AAVP particles asin the internalization assay. The medium was replaced at 48 hrs. At day4 and day 12, the culture supernatant was collected to measuresecretable cytokine levels by ELISA (Invitrogen, Carlsbad, Calif.).

Tissue Factor (TF) Assay. To examine whether secreted TNF-α/EMAP-II isfunctional, we examined its ability to induce TF synthesis inendothelial cells ECs. Briefly, 2×10⁵ HUVEC were plated on 6-well tissueculture plates per well. On the following day, cells were treated withM21 culture supernatants for 6 hrs in serum-free RPMI media. The cellswere washed with PBS and incubated with 25 mM Tris pH 7.5 for 10 min atroom temperature. The culture plates were then incubated at −80° C. for2 hrs. The total cell lysates were prepared in tissue factor assaybuffer (20 mM Tris pH 7.5, 150 mM NaCl and 0.1% BSA). Lysates werecleared by centrifugation at 13,000 rpm for 10 min. The 100 μl lysatewas analyzed for presence of tissue factor by measuring the timerequired for coagulation of Factor VIII-deficient plasma (Geroge KingBiomedical Inc, Overland Park, Kans.) in the presence of CaCl₂ (Sigma,St. Louis, Mo.) in an Amelung KC 4A Micro Coagulation Analyzer (Sigma,St. Louis, Mo.). The time required to coagulate Factor VIII-deficientplasma was converted to tissue factor units by using a standardcalibration curve plotted with known tissue factor concentrations.

In vivo AAVP Delivery and Detection by Immunofluorescence Staining. Allanimal experiments were conducted according to protocols approved by theNIH Animal Care and Use Committee. Female athymic nude mice wereobtained from the Jackson Laboratories and housed in the National CancerInstitutes, animal facility. Human melanoma cells (4×10⁶) wereinoculated subcutaneously into the right flank of nude mice. Tumorvolume (mm³) was measured in three dimensions and calculated aslength×width×height×0.52. When tumor volumes reached around 100-150 mm³,1×10¹¹ AAVP particles were injected intravenously (via tail vein).Animals were euthanized at various time intervals. Resected tumortissues and control tissues (kidney and liver) were frozen for furtheranalysis.

To detect the presence of AAVP, 5 μM thick frozen sections were stainedusing dual IF staining Briefly, sections were fixed in 4%paraformaldehyde for 5 min, followed by 2 washes in PBS for 10 min.Permeabilization was done in PBS containing 1% Triton-X-100 for 10 min.Sections were incubated with Image-iT FX signal enhancer for 30 min atroom temperature (RT), followed by three washes in PBS containing 1%Triton-X-100. Non-specific binding was blocked using 5% goat serum for30 min at RT. The primary antibodies were applied overnight at 4° C.:1:2000 dilution of anti-fd bacteriophage antibody (Sigma, St. Louis,Mo.) and 1:50 dilution of anti mouse CD31 (BD Biosciences, San Jose,Calif.). Sections were washed thrice in PBS containing 1% Triton-X-100for 10 min, then incubated with the secondary antibodies (Invitrogen,Carlsbad, Calif.): 1:400 dilutions each of goat anti-rabbit Alexa Fluor594 and goat anti-rat Alexa Fluor 488 for 30 min in the dark. Sectionswere washed thrice in PBS containing 1% Triton-X-100 for 10 min and oncewith PBS, followed by mounting in Vectashield mounting medium with DAPI(Vector Labs, Burlingame, Calif.).

TNF-α Expression In-vivo. To detect TNF-α protein expression, total celllysate was prepared from 5 μM frozen tissue sections using lysis buffer(50 mM Tris pH 7.4, 140 mM NaCl, 0.1% SDS, 1% NP40 and 0.5% sodiumdeoxycholate) containing protease inhibitor cocktail (Roche, Branchburg,N.J.). The lysates were cleared by centrifugation at 13,000 rpm for 10min. The amount of protein was quantitated using protein assay reagentfrom BioRAD. The amount of lysate equivalent to 50 μg of total proteinwas assayed for human TNF-α by ELISA (Invitrogen, Carlsbad, Calif.).

To examine localization of TNF-α expression, 5 μM frozen tissues werestained as follows. Briefly, sections were fixed in PBS containing 4%paraformaldehyde for 20 min, washed with PBS three times for 5 min eachand non-specific binding was blocked with 5% goat serum for 20 min.Sections were incubated either with 1:200 diluted anti-fd antibody(Sigma, St. Louis, Mo.) or 1:100 diluted TNF-α antibody (NovusBiologicals, Littleton, Colo.), or rat anti-mouse CD31 (BD Biosciences,San Diego, Calif.) for 1 hr., followed by three washes of 5 min each inwashing buffer (PBS containing 50 mM Tris pH 7.6 and 0.02% Tween-20).Endogenous peroxidase was blocked with 3% hydrogen peroxide for 5 minfollowed by washing and incubation with 1:200 dilution of the secondaryantimouse-HRP or biotinylated donkey anti-rat antibody for 30 min.Sections were developed with diaminobenzidine tetrahydrochloridesubstrate (Dako, Carpinteria, Calif.) for 5 min and counterstained withhematoxylin for 30 sec, rinsed in tap water, dehydrated, cleared andmounted.

Apoptosis Assay. Apoptosis was detected using the In Situ ApoptosisDetection kit, TACS TdT (R&D Systems, Minneapolis, Minn.), according tothe manufacture's recommendations.

Tumor Growth Analysis. Human melanoma cells (3×10⁶) were inoculatedsubcutaneously into the right flank of nude mice. When tumor volumesreached approximately 100 mm³, 1×10¹¹ AAVP particles were administeredvia tail vein. AAVP particle administration was repeated once more after7 days. The tumor bearing mice were followed thereafter by measuringtumor volumes every third day in a blinded fashion.

Statistical Analysis. Groups were compared by using Analysis of Variance(ANOVA) and Tukey comparison post test (GraphPad Instat Software, Inc.,San Diego, Calif.). P values <0.05 were considered statisticallysignificant.

B. Results

AAVP Particles are Internalized by Mammalian Cells. Previous studieshave shown that phage particles can be internalized by integrin-mediatedreceptor internalization (Hajitou et al., 2006). M21 cells express αvβ3receptors on their cell surface (data not shown). In order to examine,if M21 cells can internalize AAVP particles, we infected cells with AAVPparticles and then counted internalized phage. After infection, theentire extracellular virus was inactivated by subtilisin treatment,cells were lysed and internalized phage was recovered in the lysate. Theinternalized AAVP concentration was measured as TU (Table 6). There wasminimal AAVP internalization by M21 cells infected with eithernon-targeted null (fd) or TNF-α expressing non-targeted virus (fdTNF-α)in contrast to cells infected with targeted null (RGD) or targeted TNF-αexpressing AAVP (RGDTNF-α).

TABLE 6 Phage Internalization Assay Total number of phage Agent pfu/μlparticles (10²) PBS 0 0 fd  3 × 10² 1500 RGD 140 × 10² 70,000 fdTNF-α  6× 10² 3000 RGDTNF-α 215 × 10² 107,500

IF was used to visualize the localization of internalized AAVP particlesinside M21 cells. Cells infected with non-targeted TNF-α expressing AAVP(fdTNF-α) did not show phage localization (FIG. 12A, upper panel). Incontrast, cells infected with targeted AAVP expressing TNF-α (RGDTNF-α)showed enhanced localization inside M21 cells (FIG. 12A, lower panel).

AAVP Mediated Gene Expression. After determining that targeted AAVP caninfect and localize inside the mammalian cells, we investigated whethervirus infection could lead to expression of the gene product. M21 cellswere infected with AAVP expressing TNF-α, in duplicates, and productionof the TNF-α gene product was measured by ELISA. The gene product issecretable and can be detected in the culture supernatants (FIG. 12B).The supernatant tested after 5 days of infection showed TNF-α levels of800 pg/ml. The gene product tested at day 12 was higher than day 4. Thediluent control (PBS), non-targeted empty (fd), non-targeted virusexpressing TNF-α (fdTNF-α) and targeted null AAVP (RGD) had nodetectable levels of TNF-α secretion (FIG. 12B).

To examine whether secreted TNF-α by AAVP infected M21 cells wasfunctional; we tested its ability to induce tissue factor (TF) synthesisin ECs. The secreted TNF-α was capable of inducing TF expression in ECs(FIG. 12C). Recombinant TNF-α was used as a positive control. The TFinduction could be blocked by incubating culture supernatant with aTNF-α monoclonal antibody. The culture supernatant analyzed 23 dayspost-infection also showed functional TNF-α secretion (FIG. 12C). Thus,a single infection with AAVP resulted in the production of functionalgene product up to 23 days following infection.

In Vivo Tumor Targeting by AAVP. We observed AAVP infection of mammaliancells and functional expression of the TNF-α gene product in vitro. Inorder to evaluate in vivo targeting of AAVP, virus was injectedsystemically via tail vein in tumor-bearing nude mice.

Mice injected with either a diluent (PBS) or RGDTNF-α AAVP, wereeuthanized at 15 min, 1 day, 2 days, 3 days, 4 days, 8 days and 10 daysafter injection. The frozen sections from tumor tissues were analyzedfor the presence of viral particles by dual IF staining. As shown inFIG. 13, AAVP particles stain red (Alexa Flour 594), blood vessels green(Alexa Flour 488) and DAPI shows nuclear staining None of the animalsinjected with PBS showed presence of AAVP at any time point. Arepresentative tumor section from an animal injected with PBS for 15 minis shown (FIG. 13A). The animals injected with AAVP expressing RGDTNF-αshowed colocalization of virus particles in the blood vessels (FIG.13B-H). The greatest accumulation of virus particles was detected inanimals injected after 15 min (FIG. 13B). The presence of AAVP wasdetected in all the time points tested, day 1 (FIG. 13C)]], day 2 (FIG.13D), day 3 (FIG. 13E), day 4 (FIG. 13F), day 8 (FIG. 13G), and day 10(FIG. 13H). However, we noted a gradual decrease in detectable virusparticles over time.

AAVP do not Target Normal Tissues In-Vivo. In order to examine thespecificity of AAVP targeting to tumor blood vessels in vivo, weexamined two control tissues from nude mice injected with either PBS orAAVP expressing RGDTNF-α at different time points (FIG. 14 and FIG. 15).The presence of virus was detected using dual IF staining as previouslydescribed.

As shown in FIG. 14, we observed some staining of virus particles at day1 (FIG. 14A) in the liver tissue. However, the virus staining wasreduced to minimal levels by day 2 (FIG. 14B) with no virus stainingobserved at day 3 (FIG. 14C), day 8 (FIG. 14D) and day 10 (FIG. 14E).FIG. 15 shows kidney sections stained for presence of virus particles.AAVP particles were not detected in kidney in any of the time pointstested, day 1 (FIG. 15A), day 2 (FIG. 15B), day 3 (FIG. 15C), day 8(FIG. 15D) and day 10 (FIG. 15E). Nevertheless, all the kidney tissuesat different time points showed good vessels staining.

In vivo Expression of Functional Gene Product by AAVP. To examine if theAAVP particles targeted to tumor vasculature can express the geneproduct, we analyzed, in duplicates, day 3, day 4, day 8 and day 10tumor tissues for TNF-α protein levels by ELISA (FIG. 16). We observedbasal levels of endogenous TNF-α expression in all the tissues tested.Animals injected with PBS did not show any increase over the endogenouslevels at any tested time points. Mice injected with RGDTNF-α AAVPshowed TNF-α expression starting at day 4 and gradually increasing up today 10 (FIG. 16).

To determine the cell type where the gene product was being produced, westained tissue sections using an antibody specific to TNF-α. We observedthe presence of TNF-α staining around the blood vessels (FIG. 17A). Tosee the effect of TNF-α expression we stained tumor sections, forapoptosis. The DNA fragmentation was detected using TACS blue label. Theapoptotic cells stain blue (FIG. 17B, left panel). To discriminateapoptotic cells from necrotic cells, the samples were counterstainedwith nuclear fast red to aid in the morphological verification ofapoptosis. We observed blood vessels and surrounding tumor cellsundergoing apoptosis. The right panel shows blood vessels stained with aCD31 specific antibody (FIG. 17B, right panel).

Tumor Growth Analysis. We analyzed the effect of AAVP on tumorxenografts grown in nude mice in two different tumor models, TNF-αsensitive (M21) and TNF-α resistant (Pmel), to examine treatmentefficacy of AAVP expressing TNF-α. Human melanoma M21 tumors, which aresensitive to TNF-α, were grown subcutaneously in nude mice. After tumordevelopment, mice were treated systemically via tail vein injectionswith various AAVP constructs or PBS. The animals were followed for 27days. The treatment of the M21 tumors with targeted AAVP expressingTNF-α (RGDTNF-α) showed characteristic central tumor necrosis and tumorshrinkage. On day 27, the final measured time point for the differentcohorts, the PBS-treated group had mean tumor volume of 743±383 (±SD)mm³, non-targeted fdTNF group had mean tumor volume of 613±155 (±SD)mm³, null-targeted RGD phage had mean tumor volume of 622±141 (±SD) mm³and targeted TNFα expressing group had mean tumor volume of 358±98 (±5D)mm³ (p<0.048) (FIG. 18A). The reduction in tumor volume in the RGDTNF-αgroup was statistically significant starting at day 20.

In a TNF-α-resistant tumor model, human melanoma Pmel tumors weresensitized to the TNF-α effect by pre-treating them with AAVP expressingEMAP-II before administrating TNF-α. In a previous study, wedemonstrated that viral vector delivery of EMAP-II can sensitizeresistant tumors to the effects of systemically delivered TNF-α. Tumorsgrown subcutaneously in nude mice were treated systemically with AAVPexpressing EMAP-II, followed by systemic treatment with recombinantTNF-α (rTNF-α.). Tumors were followed for 2 weeks after treatment. Asshown in the FIG. 18B, mice treated with non-targeted AAVP expressingEMAP-II (fdEMAP) showed similar tumor growth compared to mice treatedwith PBS alone. Mice treated with either rTNF-α or targeted AAVPexpressing EMAP-II (RGDEMAP-II) alone showed very little effect, andwere not significantly different than the PBS or fdEMAP-II group.However, mice treated with a combination of the RGD-EMAP-II virus andrTNF-α showed significant reduction in tumor volume (p=0.007).

Example 3 Human Transferrin Peptide Targets the Transferrin/TransferrinReceptor System

Transduction of human glioma cells in culture by CRTIGPSVC (SEQ ID NO:1)AAVP—Luc U87 human-derived glioma cells were seeded onto 24 wells plateat the concentration of 40,000 cells/well and cultured O.N at 37° C.Next day, cells were incubated with AAVP, according with Nature Method'sprotocol. RGD-4C AAVP GFP and RGD-4C AAVP Luc wer used as positivecontrol for transduction efficiency. The images were taken at day 7.Phage uptake is low but increases dramatically when cells are culturedin Iron AAVP hydrogel (last column of the plate and graphic) (FIGS.19A-19B). We evaluated the specificity and efficiency of the CRTIGPSVC(SEQ ID NO:1) AAVP—Luc after systemic administration into animalsbearing U87 human—derived glioblastoma cells. Luciferase activity wasmeasured after 7 days of phage injection.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   Alauddin et al., J. Labelled Compds. Radiopharm. 46, 285-289,    (2003).-   Arap et al., Science 279, 377-380, (1998).-   Arap et al., Cancer Cell 6, 275-284, (2004).-   Arcone et al., Nucleic Acids Res., 16(8):3195-3207, 1988.-   Baichwal and Sugden, In: Gene Transfer, Kucherlapati (ed.), NY,    Plenum Press, 117-148, 1986. Barbas et al., Phage Display: A    Laboratory Manual (New York: Cold Spring Harbor Press), (2001).-   Barrow and Soothill, Trends Microbiol. 5, 268-271, (1997).-   Blasberg and Tjuvajev, J. Clin. Invest. 1 11, 1620-1 629, (2003).-   Brooks et al., Cell 79, 1157-1 164, (1994).-   Chen et al., Chem. Biol. 11, 1081-1091, (2004).-   De and Gambhir, FASEB J. 19, 2017-2019, (2005).-   Ellerby et al., Nature Med. 5, 1032-1 038, (1999).-   Geier et al., Nature 246, 221-223, (1973).-   Gelovani and Blasberg, Cancer Cell 3, 327-332, (2003).-   Gilliland et al., Proc. Natl. Acad. Sci. USA, 77(8):4539-4543, 1980.-   Giordano et al., Nature Med. 11, 1249-1253, (2001).-   Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89(12):5547-5551,    1992.-   Gross and Piwnica-Worms, Cancer Cell 7, 5-15, (2005a).-   Gross and Piwnica-Worms, Methods Enzymol. 399,5 12-530, (2005b).-   Hajitou et al., Cancer Res. 61, 3450-3457, (2001).-   Hajitou et al., Trends Cardiovasc. Med. In press, (2006).-   Hamel et al., Cancer Res. 56, 2697-2702, (1996).-   Hara et al., Gene Ther. December; 2(10):784-8, (1995)-   Hermonat and Muzyczka, Proc. Nat'l. Acad. Sci. USA 8 1:6466-6470,    (1984).-   Hood et al., Science 296, 2404-2407, (2002).-   Hsiao et al., Dev. Dyn. 220, 323-336, (2001).-   Hwu et al., J Immunol 151:4104-15, (1993)-   Ivanenkov et al., Targeted delivery of multivalent phage display    vectors into mammalian cells. Biochim. Biophys. Acta 1448, 463-472,    (1999).-   Kageyama et al, J. Biol. Chem., 262(5):2345-2351, 1987.-   Kootstra and Verma, Annu. Rev. Pharmacol. Toxicol. 43, 413-439,    (2003).-   Larocca et al., FASEB J. 13, 727-734., (1999).-   Lebkowski et al., Mol. Cell. Biol. 8:3988-3996, (1988).-   Lieber et al., J. Virol. 73,93 14-9324, (1999).-   Luker et al., PNAS 10 1, 12288-12293, (2004).-   Macejak and Sarnow, Nature, 353:90-94, 1991.-   Machida, Viral Vectors for Gene Therapy (Totowa, N.J.: Humana    Press), (2003).-   Marchib et al., Cancer Cell 5, 15 1-162, (2004).-   Massoud and Gambhir, Genes Dev. 17, 545-580, (2003).-   McCarty et al., Annu Rev. Genet. 38, 819-845, (2004).-   Miller et al., Nature Biotechnol. 21, 1040-1046, (2003).-   Mizuguchi and Hayakawa, Hum. Gene Ther. 15, 1034-1044, (2004).-   Muzyczka, Curr. Top. Microbiol. Immunol. 158:97-129, (1992).-   Nakamura et al., Hum. Gene Ther. 13, 613-626, (2002).-   Nicolas and Rubenstein, In: Vectors: A survey of molecular cloning    vectors and their uses, Rodriguez and Denhardt (Eds.), Stoneham:    Butterworth, 493-513, 1988.-   Oliviero et al., EMBO J., 6(7):1905-1912, 1987.-   Pasqualini et al., Nature Biotechnol. 15, 542-546, (1997).-   Pelletier and Sonenberg, Nature, 334:320-325, 1988.-   Piersanti et al., J. Mol. Med. 82, 467-476, (2004).-   Poul and Marks, J. Mol. Biol. 288, 203-21 1, (1999).-   Prowse and Baumann, Mol. Cell. Biol., 8(1):42-51, 1988.-   Ray et al., Semin. Nucl. Med. 3 1, 3 12-320, (2001).-   Remington's Pharmaceutical Sciences, 15th ed., pages 1035-1038 and    1570-1580, Mack Publishing Company, Easton, Pa., 1980.-   Ridgeway, In: Vectors: A survey of molecular cloning vectors and    their uses, Stoneham: Butterworth, 467-492, 1988.-   Ron et al., Mol Cell Endocrinol. 21; 74 (3):C97-104,1990.-   Sambrook et al., In: Molecular cloning, Cold Spring Harbor    Laboratory Press, Cold Spring Harbor, N.Y., 2001.-   Spanjer and Scherphof, Biochim Biophys Acta.; 734(1):40-7, 1983-   Shayakhmetov et al., J. Virol. 79, 7478-749 1, (2005).-   Sipkins et al., Nature Med. 4, 623-626, (1998).-   Souza et al., PNAS 103, 1215-1220,2006, (2006).-   Tai and Laforest, Annu Rev. Biomed. Eng. 7, 255-285, (2005).-   Tandle et al., Cytokine 30:347-58, (2005)-   Temin, In: Gene Transfer, Kucherlapati (ed.), NY, Plenum Press,    149-188, 1986.-   Tjuvajev et al., Cancer Res. 58, 4333-4341, (1998).-   Tjuvajev et al., Cancer Res. 59,5 186-5193, (1999).-   Tratschin et al., Mol. Cell. Biol. 5(11):3251-3260, (1985).-   Trepel et al., Cancer Res. 61, 8 1 10-81 12, (2001).-   Uhrbom et al., Nature Med. 10, 1257-1 260, (2004).-   Wagner et al., PNAS 89:7934-7938, (1992).-   Walensky et al., Science 305, 14 1 1-14 13, (2004).-   White et al., Circulation 109, 5 13-5 19, (2004).-   Wilson et al., Mol. Cell. Biol., 10(12):6181-6191, 1990.-   Xiao et al., J. Virol. 72, 2224-2232, (1998).-   Yang et al., J. Virol. 7 1, 923 1-9247, (1997).-   Zacher et al., Gene 9, 127-140, (1980).-   Zechner et al., Mol. Cell. Biol., 8(6):2394-2401, 1988.

1-50. (canceled)
 51. A method of detecting gene transfer in a subject,comprising: (a) administering a therapeutic adeno-associated viral/phagevector (AAVP) encoding a reporter to a subject having, suspected ofhaving or at risk of developing a pathologic or disease condition; and(b) evaluating in situ expression of the therapeutic AAVP in a tissue orcell targeted for treatment by detecting the encoded reporter orreporter activity.
 52. The method of claim 1, further comprisingadministering a cancer treatment to the subject based on expression of atherapeutically sufficient level of a therapeutic gene expressed by theAAVP nucleic acid in the target organ, tissue or cell.
 53. The method ofclaim 2, wherein a second therapeutic AAVP is administered if theexpression of the first therapeutic AAVP is not expressed at atherapeutically effective level.
 54. The method of claim 1, whereinevaluation of AAVP expression is by non-invasive detection of thereporter or an activity of the reporter.
 55. The method of claim 4,wherein the reporter is an enzyme.
 56. The method of claim 55, whereinthe enzyme is a kinase.
 57. The method of claim 56, wherein the kinaseis thymidine kinase.
 58. The method of claim 57, wherein the kinasemodifies a compound labeled with a detectable label.
 59. The method ofclaim 58, wherein the detectable label is detectable by fluorescence,chemiluminescence, surface enhanced raman spectroscopy (SERS), magneticresonance imaging (MRI), computer tomography (CT), or positron emissiontomography (PET) imaging.
 60. The method of claim 58, wherein thedetectably labeled compound is a nucleoside analog.
 61. The method ofclaim 60, wherein the detectably labeled compound is fluorodeoxyglucose(FDG); 2′-fluoro-2′deoxy-1beta-D-arabionofuranosyl-5-ethyl-uracil(FEAU); 5-[¹²³I]-2′-fluoro-5-iodo-1β-D-arabinofuranosyl-uracil;5-[¹²⁴I]-2′-fluoro-5-iodo-1β-D-arabinofuranosyl-uracil;5-[¹³¹I]-2′-fluoro-5-iodo-1β-D-arabinofuranosyl-uracil,5-[¹⁸F]-2′-fluoro-5-fluoro-1-β-D-arabinofuranosyl-uracil; 2-[¹¹I]- and5-([¹¹C]-methyl)-2′-fluoro-5-methyl-1-β-D-arabinofuranosyl-uracil;2-[¹¹C]-2′-fluoro-5-ethyl-1-β-D-arabinofuranosyl-uracil;5-([¹¹C]-ethyl)-2′-fluoro-5-ethyl-1-β-D-arabinofuranosyl-uracil;5-(2-[¹⁸F]-ethyl)-2′-fluoro-5-(2-fluoro-ethyl)-1-β-D-arabinofuranosyl-uracil,5-[¹²³I]-2′-fluoro-5-iodovinyl-1-β-D-arabinofuranosyl-uracil;5-[¹²⁴I]-2′-fluoro-5-iodovinyl-1-β-D-arabinofuranosyl-uracil;5-[¹³¹I]-2′-fluoro-5-iodovinyl-1-β-D-arabinofuranosyl-uracil;5-[¹²³I]-2′-fluoro-5-iodo-1-β-D-ribofuranosyl-uracil;5-[¹²⁴I]-2′-fluoro-5-iodo-1-β-D-ribofuranosyl-uracil;5-[¹³¹I]-2′-fluoro-5-iodo-1-β-D-ribofuranosyl-uracil;5-[¹²³I]-2′-fluoro-5-iodovinyl-1-β-D-ribofuranosyl-uracil;5-[¹²⁴I]-2′-fluoro-5-iodovinyl-1-β-D-ribofuranosyl-uracil;5-[¹³¹I]-2′-fluoro-5-iodovinyl-1-β-D-ribofuranosyl-uracil; or9-4-[¹⁸F]fluoro-3-(hydroxymethyl)butyl]guanine.
 62. The method of claim58, wherein the detectably labeled compound comprises a ¹⁸F, ²⁷⁷Ac,²¹¹At, ¹²⁸Ba, ¹³¹Ba, ⁷Be, ²⁰⁴Bi, ²⁰⁵Bi, ²⁰⁶Bi, ⁷⁶Br, ⁷⁷Br, ⁸²Br, ¹⁰⁹Cd,⁴⁷Ca, ¹¹C, ¹⁴C, ³⁶Cl, ⁴⁸Cr, ⁵¹Cr, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ¹⁶⁵Dy, ¹⁵⁵Eu, ¹⁵³Gd,⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁷²Ga, ¹⁹⁸Au, ³H, ¹⁶⁶Ho, ¹¹¹In, ¹¹³In, ¹¹⁵In, ¹²³I,¹²⁵I, ¹³¹I, ¹⁸⁹Ir, ¹⁹¹Ir, ¹⁹²Ir, ¹⁹⁴Ir, ⁵²Fe, ⁵⁹Fe, ¹⁷⁷Lu, ¹⁵O, ¹⁹¹Os,¹⁰⁹Pd, ³²P, ³³P, ⁴²K, ²²⁶Ra, ¹⁸⁶Re, ¹⁸⁸Re, ⁸²Rb, ¹⁵³Sm, ⁴⁶Sc, ⁷²Se,⁷⁵Se, ¹⁰⁵Ag, ²²Na, ²⁴Na, ⁸⁹Sr, ³⁵S, ³⁸S, ¹⁷⁷Ta, ⁹⁶Tc, ^(99m)Tc, ²⁰¹Tl,²⁰²Tl, ¹¹³Sn, ^(117m)Sn, ¹²¹Sn, ¹⁶⁶Yb, ¹⁶⁹Yb, ¹⁷⁵Yb, ⁸⁸Y, ⁹⁰Y, ⁶²Zn, or⁶⁵Zn.
 63. The method of claim 58, wherein the detectable label is ¹³¹I,¹²⁵I, ¹²³I, ¹¹¹I, ^(99m)Tc, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ³²P, ¹⁵³Sm, ⁶⁷Ga, ²⁰¹Tl,⁷⁷Br, or ¹⁸F label.
 64. The method of claim 51, wherein the AAVPcomprises a moiety that selectively targets a tissue or cell targetedfor treatment.
 65. The method of claim 64, wherein the moiety is encodedby a capsid protein of the AAVP.
 66. The method of claim 65, wherein thecapsid protein is recombinant capsid protein.
 67. The method of claim66, wherein the recombinant capsid protein comprises a targetingpeptide.
 68. The method of claim 67, wherein the targeting peptide is acyclic peptide.
 69. The method of claim 67, wherein the targetingpeptide is a linear peptide.
 70. The method of claim 67, wherein thetargeting peptide selectively binds a cell expressing an integrin on thecell surface.
 71. The method of claim 70, wherein the integrin is αvβ3or αvβ5 integrin.
 72. The method of claim 67, wherein the targetingpeptide comprises an RGD motif.
 73. The method of claim 19, wherein thetargeting peptide selectively binds a cell expressing a transferrinreceptor.
 74. The method of claim 73, wherein the peptide comprises anamino acid sequence comprising CRTIGPSVC.
 75. The method of claim 51,wherein the subject has, is suspected of having, or at risk ofdeveloping a hyperproliferative disease.
 76. The method of claim 75,wherein the hyperproliferative disease is fibrosarcoma, myosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer,pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer,cancer of the head and neck, skin cancer, brain cancer, squamous cellcarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcancer, testicular cancer, small cell lung carcinoma, non-small celllung carcinoma, bladder carcinoma, epithelial carcinoma, glioma,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposisarcoma
 77. The method of claim 51, wherein the reporter gene isoperatively coupled a tissue or cell specific promoter.
 78. The methodof claim 51, wherein evaluating expression comprises administering alabel that is selectively metabolized by a cell expressing the AAVPnucleic acid.
 79. The method of claim 51, wherein the therapeutic AAVPencodes a second therapeutic gene.
 80. The method of claim 79, whereinthe second therapeutic gene is tumor suppressor, inhibitory RNA,inhibitory DNA, or prodrug converting enzyme.